Non-platelet depleting and non-red blood cell depleting cd47 antibodies and methods of use thereof

ABSTRACT

This invention relates generally to monoclonal antibodies that recognize CD47, more specifically to CD47 antibodies that do not cause a significant level of agglutination of cells, red blood cell depletion, anemia, and/or platelet depletion, to methods of generating these antibodies, and to methods of using these monoclonal antibodies as therapeutics.

RELATED APPLICATIONS

This application is a continuation patent application of U.S.application Ser. No. 14/841,619, filed Aug. 31, 2015 which is acontinuation-in-part of U.S. application Ser. No. 13/761,087, filed Feb.6, 2013 and a continuation-in-part of PCT Application No.PCT/US2013/024995, filed Feb. 6, 2013, both of which claim the benefitof, and priority to, U.S. Provisional Application No. 61/595,216, filedFeb. 6, 2012, and U.S. Provisional Application No. 61/659,752, filedJun. 14, 2012. This application also claims the benefit of, and priorityto, U.S. Provisional Application No. 61/815,219, filed Apr. 23, 2013.The contents of each of these applications are incorporated herein byreference in their entireties.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 683772000301SeqList.txt,date recorded: Mar. 11, 2016, size: 90 KB).

FIELD OF THE INVENTION

This invention relates generally to monoclonal antibodies that recognizeCD47, more specifically to CD47 antibodies that do not cause asignificant level of hemagglutination of human red blood cells, redblood cell depletion, anemia, and/or platelet depletion, to methods ofgenerating these antibodies, and to methods of using these monoclonalantibodies as therapeutics.

BACKGROUND OF THE INVENTION

CD47, also known as integrin-associated protein (IAP), ovarian cancerantigen OA3, Rh-related antigen and MER6, is a multi-spanningtransmembrane receptor belonging to the immunoglobulin superfamily. CD47expression and/or activity have been implicated in a number of diseasesand disorders. Accordingly, there exists a need for therapies thattarget CD47. In addition, due to expression of CD47 on platelets, thereis also a need for CD47-targeting therapies (e.g., antibodies) that donot cause significant levels of platelet depletion, hemagglutination,red blood cell depletion, and/or anemia when administered to a subject.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies that recognize andbind to CD47, particularly human CD47. The antibodies of the inventionare capable of modulating, e.g., blocking, inhibiting, reducing,antagonizing, neutralizing or otherwise interfering with CD47expression, activity and/or signaling, and these antibodies do not causea significant level of hemagglutination of human red blood cells, alsoreferred to herein as erythrocytes. However, the ability of theantibodies of the present invention to bind CD47 on the cell surface andnot cause a cellular clumping phenomenon is not limited to red bloodcells. The antibodies of the present invention uniquely bind CD47 in amanner that does not promote clumping of CD47 positive cells. Inaddition or alternatively, the antibodies of the present invention donot cause significant depletion of platelets upon administration. Theantibodies of the invention and derivatives thereof are capable ofmodulating, e.g., blocking, inhibiting, reducing, antagonizing,neutralizing or otherwise interfering with the interaction between CD47and SIRPα (signal-regulatory-protein a), and these antibodies do notcause a significant level of hemagglutination of human red blood cells.The antibodies provided herein are referred to collectively as “CD47antibodies.” The CD47 antibodies of the invention are a significantimprovement over existing CD47 antibodies that cause hemagglutination ofhuman red blood cells (See, e.g., Kikuchi Y, Uno S, Yoshimura Y et al. Abivalent single-chain Fv fragment against CD47 induces apoptosis forleukemic cells. Biochem Biophys Res Commun 2004; 315: 912-8). Forexample, the CD47 antibodies of the invention are a significantimprovement over the existing CD47 antibodies B6H12, BRC126, and CC2C6,each of which block SIRPα, but cause hemagglutination of RBCs, asdescribed in detail below. For example, the CD47 antibodies of theinvention are a significant improvement over an affinity-evolvedSIRPα-Fc fusion protein that, when administered to mice and/orcynomolgus monkeys, caused red blood cell loss and anemia (See, Weiskopfet al. Engineered SIRPa Variants as Immunotherapeutic Adjuvants toAnticancer Antibodies. Science 2013; 341:88). The full IgG CD47antibodies of the present invention (e.g., 2A1 and its humanizedderivatives including those provided in Table 1) do not agglutinatecells at a significant level. For example, the CD47 antibodies of theinvention do not hemagglutinate red blood cells (RBCs). Described hereinare CD47 antibodies in a full IgG format that block SIRPα and do notcause a significant level of agglutination and/or platelet depletion. Inaddition, the CD47 antibodies of the invention do not cause asignificant level of RBC depletion and/or anemia.

The CD47 antibodies of the invention exhibit numerous desirablecharacteristics, such as, by way of non-limiting example, potentblocking of the interaction between CD47 and its ligand SIRPα, withoutcausing a significant level of hemagglutination of erythrocytes, as wellas potent anti-tumor activity. For example, the CD47 antibodies of theinvention block at least 40%, at least 45%, at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 95%, or at least 99% of the interaction between CD47and SIRPα as compared to the level of interaction between CD47 and SIRPαin the absence of the CD47 antibody described herein.

The CD47 antibodies of the invention do not cause a significant level ofagglutination of cells, e.g., the CD47 antibodies of the invention donot cause a significant level of hemagglutination of red blood cells. Insome cases, a significant level of agglutination of cells refers to thelevel of agglutination in the presence of existing CD47 antibodies. Inone aspect, the level of agglutination in the presence of the CD47antibodies of the invention is reduced by at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 99% compared to thelevel of agglutination in the presence existing CD47 antibodies. In someembodiments, the CD47 antibodies of the invention do not cause asignificant level of agglutination if the level of agglutination in thepresence of the CD47 antibodies of the invention is reduced by at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least99% compared to the level of agglutination in the presence of existingCD47 antibodies. In other embodiments, the CD47 antibodies of theinvention do not cause a significant level of agglutination if the levelof agglutination in the presence of the CD47 antibodies of the inventionis reduced by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 99% compared to the level of agglutination in thepresence of CD47 antibody, 1B4, which comprises a variable heavy andvariable light chain sequence provided in SEQ ID NO: 80 and SEQ ID NO:81, respectively. Preferably, the CD47 antibodies of the invention donot cause a significant level of agglutination of cells at an antibodyconcentration of between 10 pM and 10 μM, e.g., at an antibodyconcentration of 50 pM, 100 pM, 1 nM, 10 nM, 50 nM, 100 nM, 1 μM, or 5μM.

In some embodiments, the level of RBC depletion is determined bymeasuring the RBC count in a subject after administration of atreatment, e.g., an antibody of the invention. In some embodiments, theCD47 antibodies of the invention do not cause a significant level of RBCdepletion if the RBC count in a subject after administration of anantibody of the invention is within the range of a normal, healthysubject. For example, the RBC count for a normal, healthy male human isabout 4.7 to about 6.1 million cells per microliter of blood sample. Forexample, the RBC count for a normal, healthy female human is 4.2 toabout 5.4 million cells per microliter of blood sample. In someembodiments, the CD47 antibodies of the invention do not cause asignificant level of RBC depletion if the RBC count in a subject afteradministration (5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 12 h, 24h, 2 days, 4 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months,or more) of an antibody of the invention is at least 50%, 60%, 70%, 80%,90%, 95%, 97%, 98%, 99%, or 99.5% of the RBC count prior toadministration. Alternatively or in addition, the CD47 antibodies of theinvention do not cause a significant level of RBC depletion if the RBCcount in a subject after administration (5 min, 10 min, 30 min, 1 h, 2h, 3 h, 4 h, 5 h, 12 h, 24 h, 2 days, 4 days, 6 days, 1 week, 2 weeks, 3weeks, 1 month, 2 months, or more) of an antibody of the invention is atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the RBCcount in a subject after administration of a placebo treatment (e.g.,vehicle). RBC counts are determined by standard methods in the art.Preferably, the CD47 antibodies of the invention do not cause asignificant level of RBC depletion at an antibody concentration ofbetween 10 pM and 10 μM, e.g., at an antibody concentration of 50 pM,100 pM, 1 nM, 10 nM, 50 nM, 100 nM, 1 μM, or 5 μM. In some embodiments,the CD47 antibodies of the invention do not cause a significant level ofRBC depletion when administered at a dose of 0.1 mg/kg, 0.5 mg/kg, 1mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, or greater.

The CD47 antibodies of the present invention do not cause a significantlevel of platelet depletion. For example, administration of an antibodyof the invention leads to a percentage of platelets remaining of atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Preferably, the CD47antibodies of the invention do not cause a significant level of plateletdepletion at an antibody concentration of between 10 pM and 10 μM, e.g.,at an antibody concentration of 50 pM, 100 pM, 1 nM, 10 nM, 50 nM, 100nM, 1 μM, or 5 μM.

Also, the CD47 antibodies of the present invention include but are notlimited to antibodies that have a low binding affinity to a Fcγ receptor(FcγR). For example, the constant region of the antibody has a lowerbinding affinity to a FcγR than the constant region of an antibody of asubclass such as IgG1 (wild type or mutant), IgG4 (wild type or mutant,e.g., IgG4P).

The antibodies of the present invention are also significantly morepotent in tumor models compared to antibodies known in the art. Forexample, the ability of macrophages to phagocytose tumor cells in thepresence of CD47 antibodies of the invention is increased by at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least99% compared to the ability of macrophages to phagocytose tumor cells inthe presence of existing CD47 antibodies.

Those skilled in the art will recognize that it is possible toquantitate, without undue experimentation, the level of agglutination,e.g., the level of hemagglutination of RBCs. For example, those skilledin the art will recognize that the level of hemagglutination isascertained by measuring the area of an RBC dot after performing ahemagglutination assay in the presence of the CD47 antibodies of theinvention, as described in the Examples below. In some cases, the areaof the RBC dot in the presence of the CD47 antibody of the invention iscompared to the area of the RBC dot in the absence of a CD47 antibody,i.e., in the presence of zero hemagglutination. In this manner,hemagglutination is quantified relative to a baseline control. A largerRBC dot area corresponds to a higher level of hemagglutination.Alternatively, densitometry of the RBC dot may also be utilized toquantitate hemagglutination.

Those skilled in the art will recognize that it is possible toquantitate, without undue experimentation, the level of RBC depletion.For example, those skilled in the art will recognize that the level ofRBC depletion is ascertained, e.g., by measuring the RBC count (i.e.,the total number of RBCs in a sample of blood), e.g., by using a cellcounter or a hemacytometer. Those of skill in the art will recognizethat the RBCs in a sample of blood can optionally be isolated byfractionating whole blood using, e.g., centrifugation, prior tocounting. In some cases, the RBC count in the presence of an CD47antibody of the invention is compared to the RBC count in the absence ofthe CD47 antibody, i.e., in the presence of zero RBC depletion. In thismanner, the level of RBC depletion is normalized relative to a baselinecontrol.

The CD47 antibodies described herein are useful in treating, delayingthe progression of, preventing relapse of or alleviating a symptom of acancer or other neoplastic condition. For example, the CD47 antibodiesdescribed herein are useful in treating hematological malignanciesand/or tumors, e.g., hematological malignancies and/or tumors. Forexample, the CD47 antibodies described herein are useful in treatingCD47+ tumors. By way of non-limiting example, the CD47 antibodiesdescribed herein are useful in treating non-Hodgkin's lymphoma (NHL),acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiplemyeloma (MM), breast cancer, ovarian cancer, head and neck cancer,bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lungcancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and so on.Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors,pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors,lung tumors, head and neck tumors, bladder tumors, esophageal tumors,liver tumors, and kidney tumors.

As used herein, “hematological cancer” refers to a cancer of the blood,and includes leukemia, lymphoma and myeloma among others. “Leukemia”refers to a cancer of the blood in which too many white blood cells thatare ineffective in fighting infection are made, thus crowding out theother parts that make up the blood, such as platelets and red bloodcells. It is understood that cases of leukemia are classified as acuteor chronic. Certain forms of leukemia include, by way of non-limitingexample, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML);chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML);Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasiasyndrome. “Lymphoma” may refer to a Hodgkin's lymphoma, both indolentand aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, andfollicular lymphoma (small cell and large cell), among others. Myelomamay refer to multiple myeloma (MM), giant cell myeloma, heavy-chainmyeloma, and light chain or Bence-Jones myeloma.

Exemplary monoclonal antibodies of the invention include, for example,the antibodies described herein. Exemplary antibodies include antibodieshaving a variable heavy chain selected from SEQ ID NOs: 5-30 and avariable light chain selected from SEQ ID NOs: 31-47. The antibodiesalso include antibodies having a variable heavy chain that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe sequence set forth in at least one of SEQ ID NOs: 5-30 and avariable light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the sequence set forth in at leastone of SEQ ID NOs: 31-47. Preferably, the antibodies recognize and bindto human CD47 and do not cause a significant level of hemagglutinationof human red blood cells. These antibodies are respectively referred toherein as CD47 antibodies. CD47 antibodies include fully humanmonoclonal antibodies, as well as humanized monoclonal antibodies andchimeric antibodies. These antibodies show specificity for human CD47,and they have been shown to modulate, e.g., block, inhibit, reduce,antagonize, neutralize or otherwise interfere with CD47 expression,activity and/or signaling without causing a significant level ofhemagglutination of red blood cells, red blood cell depletion, anemia,and/or platelet depletion.

The CD47 antibodies provided herein exhibit inhibitory activity, forexample by inhibiting CD47 expression (e.g., inhibiting cell surfaceexpression of CD47), activity, and/or signaling, or by interfering withthe interaction between CD47 and SIRPα. The antibodies provided hereincompletely or partially reduce or otherwise modulate CD47 expression oractivity upon binding to, or otherwise interacting with, CD47, e.g., ahuman CD47. The reduction or modulation of a biological function of CD47is complete, significant, or partial upon interaction between theantibodies and the human CD47 polypeptide and/or peptide. The antibodiesare considered to completely inhibit CD47 expression or activity whenthe level of CD47 expression or activity in the presence of the antibodyis decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% ascompared to the level of CD47 expression or activity in the absence ofinteraction, e.g., binding, with the antibody described herein. The CD47antibodies are considered to significantly inhibit CD47 expression oractivity when the level of CD47 expression or activity in the presenceof the CD47 antibody is decreased by at least 50%, e.g., 55%, 60%, 75%,80%, 85% or 90% as compared to the level of CD47 expression or activityin the absence of binding with a CD47 antibody described herein. Theantibodies are considered to partially inhibit CD47 expression oractivity when the level of CD47 expression or activity in the presenceof the antibody is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%,40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of CD47expression or activity in the absence of interaction, e.g., binding,with an antibody described herein.

Antibodies of the invention also include monoclonal antibodies thatspecifically bind CD47, wherein the antibody does not cause asignificant level of agglutination, e.g., red blood cellhemagglutination (“RBC hemagglutination”). The antibodies of the presentinvention uniquely bind CD47 in a manner that does not promote clumpingof CD47 positive cells; however, the ability of the antibodies of thepresent invention to bind CD47 on the cell surface and not cause acellular clumping phenomenon is not limited to red blood cells.Additionally or alternatively, the antibodies of the present inventiondo not cause a significant level of platelet depletion, RBC depletion,and/or anemia.

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

The invention provides monoclonal antibodies that bind to CD47 or animmunologically active fragment thereof, wherein the antibody does notcause a significant level of agglutination of cells afteradministration, e.g., the antibody does not cause a significant level ofhemagglutination of red blood cells after administration. In addition oralternatively, the antibody or fragment thereof does not cause asignificant level of platelet depletion. In some embodiments, theantibody is chimeric, humanized, or fully human. In some embodiments,the antibodies bind to human CD47. In some embodiments, the antibody orimmunologically active fragment thereof prevents CD47 from interactingwith SIRPα. The antibodies are considered to completely inhibit theinteraction of CD47 and SIRPα when the level of CD47/SIRPα interactionin the presence of the antibody is decreased by at least 95%, e.g., by96%, 97%, 98%, 99% or 100% as compared to the level of CD47/SIRPαinteraction in the absence of interaction with the antibody, e.g.,binding with the antibody. The antibodies are considered to partiallyinhibit CD47/SIRPα interaction when the level of CD47/SIRPα interactionin the presence of the antibody is decreased by less than 95%, e.g.,10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared tothe level of CD47/SIRPα interaction in the absence of interaction withthe antibody, e.g., binding with the antibody.

The amount of antibody sufficient to treat or prevent cancer in thesubject is, for example, an amount that is sufficient to reduce CD47signaling (See, e.g., Yamauchi et al., 2013 Blood, January 4. [Epubahead of print]; Soto-Pantoja et al., 2013 Expert Opin Ther Targets, 17:89-103; Irandoust et al., 2013 PLoS One, Epub January 8; Chao et al.,2012 Curr Opin Immunol, 24:225-32; Theocharides et al., 2012 J Exp Med,209(10): 1883-99; Csányi et al., 2012 Arterioscler Thromb Vasc Biol, 32:2966-73; Maxhimer et al., 2009 Sci Transl Med, 1: 3ra7; Sarfati et al.,2008 Curr Drug Targets, 9: 842-850; Miyashita et al., 2004 Mol BiolCell, 15: 3950-3963; E. J. Brown and W. A. Frazier, 2001 Trends CellBiol, 11: 130-135; Oldenborg et al., 2001 J Exp Med, 193: 855-862;Blazar et al., 2001 J Exp Med, 194: 541-549; Oldenborg et al., 2000Science, 288: 2051-2054; and Gao et al., 1996 J Biol Chem, 271: 21-24).For example, the amount of antibody sufficient to treat or preventcancer in the subject is an amount that is sufficient to reduce thephagocytic inhibitory signal in macrophages generated by CD47/SIRPαinteraction in the CD47/SIRPα signaling axis, i.e., the antibody of theinvention promotes macrophage-mediated phagocytosis of a CD47-expressingcell. As used herein, the term “reduced” refers to a decreased CD47signaling in the presence of the antibody of the invention. CD47mediated signaling is decreased when the level of CD47 signaling in thepresence of a CD47 antibody of the invention is greater than or equal to5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or100% lower than a control level of CD47 signaling (i.e., the level ofCD47 signaling in the absence of the antibody). Level of CD47 signalingis measured using any of a variety of standard techniques, such as, byway of non-limiting example, measurement of down-stream gene activation,and/or luciferase reporter assays responsive to CD47 activation. Thoseskilled in the art will appreciate that the level of CD47 signaling canbe measured using a variety of assays, including, for example,commercially available kits.

In some embodiments, the antibody or immunologically active fragmentthereof is an IgG isotype. In some embodiments, the constant region ofthe antibody is of human IgG1 isotype, having an amino acid sequence:

(SEQ ID NO: 1)

In some embodiments, the human IgG1 constant region is modified at aminoacid Asn297 (Boxed, Kabat Numbering) to prevent to glycosylation of theantibody, for example Asn297Ala (N297A). In some embodiments, theconstant region of the antibody is modified at amino acid Leu235 (KabatNumbering) to alter Fc receptor interactions, for example Leu235Glu(L235E) or Leu235Ala (L235A). In some embodiments, the constant regionof the antibody is modified at amino acid Leu234 (Kabat Numbering) toalter Fc receptor interactions, e.g., Leu234Ala (L234A). In someembodiments, the constant region of the antibody is altered at bothamino acid 234 and 235, for example Leu234Ala and Leu235Ala(L234A/L235A) (EU index of Kabat et al 1991 Sequences of Proteins ofImmunological Interest).

In some embodiments, the constant region of the antibody is of humanIgG2 isotype, having an amino acid sequence:

(SEQ ID NO: 2)

In some embodiments, the human IgG2 constant region is modified at aminoacid Asn297 (Boxed, Kabat Numbering) to prevent to glycosylation of theantibody, e.g., Asn297Ala (N297A).

In some embodiments, the constant region of the antibody is of humanIgG3 isotype, having an amino acid sequence:

(SEQ ID NO: 3)

In some embodiments, the human IgG3 constant region is modified at aminoacid Asn297 (Boxed, Kabat Numbering) to prevent to glycosylation of theantibody, e.g., Asn297Ala (N297A). In some embodiments, the human IgG3constant region is modified at amino acid 435 to extend the half-life,e.g., Arg435His (R435H) (EU index of Kabat et al 1991 Sequences ofProteins of Immunological Interest).

In some embodiments, the constant region of the antibody is of humanIgG4 isotype, having an amino acid sequence:

(SEQ ID NO: 4)

In some embodiments, the human IgG4 constant region is modified withinthe hinge region to prevent or reduce strand exchange, e.g., Ser228Pro(S228P). In other embodiments, the human IgG4 constant region ismodified at amino acid 235 to alter Fc receptor interactions, e.g.,Leu235Glu (L235E). In some embodiments, the human IgG4 constant regionis modified within the hinge and at amino acid 235, e.g., Ser228Pro andLeu235Glu (S228P/L235E). In some embodiments, the human IgG4 constantregion is modified at amino acid Asn297 (Kabat Numbering) to prevent toglycosylation of the antibody, e.g., Asn297Ala (N297A). In someembodiments of the invention, the human IgG4 constant region is modifiedat amino acid positions Ser228, Leu235, and Asn297 (e.g.,S228P/L235E/N297A). (EU index of Kabat et al 1991 Sequences of Proteinsof Immunological Interest). In other embodiments of the invention, theantibody is of human IgG4 subclass and lacks glycosylation. In theseembodiments the glycosylation can be eliminated by mutation at position297 (Kabat numbering), for example N297A. In other embodiments, theglycosylation can be eliminated by production of the antibody in a hostcell that lacks the ability for post-translational glycosylation, forexample a bacterial or yeast derived system or a modified mammalian cellexpression system.

In some embodiments, the human IgG constant region is modified toenhance FcRn binding. Examples of Fc mutations that enhance binding toFcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E,respectively) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol281(33) 23514-23524), or Met428Leu and Asn434Ser (M428L, N434S)(Zalevsky et al 2010 Nature Biotech, Vol 28(2) 157-159). (EU index ofKabat et al 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the human IgG constant region is modified to alterantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), e.g., the amino acidmodifications described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al.,2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010,Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. EnzymeRegul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468;Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

In some embodiments, the human IgG constant region is modified to induceheterodimerization. For example, having an amino acid modificationwithin the CH3 domain at Thr366, which when replaced with a more bulkyamino acid, e.g., Try (T366W), is able to preferentially pair with asecond CH3 domain having amino acid modifications to less bulky aminoacids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Val,respectively (T366S/L368A/Y407V). Heterodimerization via CH3modifications can be further stabilized by the introduction of adisulfide bond, for example by changing Ser354 to Cys (S354C) and Y349to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journalof Immunological Methods, 248: 7-15).

In other embodiments of the invention, the antibody lacks glycosylation,but is not modified at amino acid Asn297 (Kabat numbering). In theseembodiments the glycosylation can be eliminated by production of theantibody in a host cell that lacks a post-translational glycosylationcapacity, for example a bacterial or yeast derived system or a modifiedmammalian cell expression system.

The invention also provides pharmaceutical compositions that include oneor more monoclonal antibodies that bind to CD47 or an immunologicallyactive fragment thereof, wherein the antibody does not cause asignificant level of hemagglutination of red blood cells afteradministration.

Hemagglutination is an example of a homotypic interaction, wherein twoCD47 expressing cells are caused to aggregate or clump when treated witha bivalent CD47 binding entity. The ability of the antibodies of thepresent invention to bind CD47 on the cell surface and not cause acellular clumping phenomenon is not limited to red blood cells. Theantibodies of the present invention have been observed to uniquely bindCD47 in a manner that does not promote clumping of CD47 positive celllines, e.g., Daudi cells.

In some cases, the antibody comprises a variable heavy (VH) chain regionselected from the group consisting of SEQ ID NOs: 5-30. The antibodyoptionally comprises a variable light (VL) chain region selected fromthe group consisting of SEQ ID NOs: 31-47. In some cases, the antibodycomprises a VH chain region selected from the group consisting of SEQ IDNOs: 5-30 and a VL chain region selected from the group consisting ofSEQ ID NOs: 31-47. The antibodies of the invention also includeantibodies having a variable heavy chain that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence setforth in at least one of SEQ ID NOs: 5-30 and a variable light chainthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the sequence set forth in at least one of SEQ ID NOs:31-47. In other aspects, the antibody comprises a VH region provided inany one of SEQ ID NOs: 5, 7, 8, 11, 15-17, 20-22, and 27-30 paired witha VL region provided in any one of SEQ ID NOs: 31-39, 42, 43, 44, and47. In another embodiment, the antibody comprises a VH region providedin any one of SEQ ID NOs: 5, 7, 8, 11, 12, 15-17, 20-22, and 27-30paired with a VL region provided in any one of SEQ ID NOs: 31, 32, 35,40, 41, 42, 43, 44, and 47. In yet another aspect, the antibodycomprises a combination of a VH chain region and a VL chain regionselected from the combinations listed in Table 1.

In some embodiments, the CD47 antibody or immunologically activefragment thereof comprises a VH complementarity determining region 1(CDR1) sequence set forth in SEQ ID NO: 50, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ IDNO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66, a VH CDR2sequence set forth in SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76, a VH CDR3 sequence set forthin SEQ ID NO: 52 or SEQ ID NO: 77, a VL CDR1 sequence set forth in SEQID NO: 53, SEQ ID NO: 67, or SEQ ID NO: 68, a VL CDR2 sequence set forthin SEQ ID NO: 54, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71 and aVL CDR3 sequence set forth in SEQ ID NO: 55. For example, the antibodyor immunologically active fragment thereof comprises a VH CDR1 sequenceset forth in SEQ ID NO: 50, a VH CDR2 sequence set forth in SEQ ID NO:51, a VH CDR3 sequence set forth in SEQ ID NO: 52, a VL CDR1 sequenceset forth in SEQ ID NO: 53, a VL CDR2 sequence set forth in SEQ ID NO:54, and a VL CDR3 sequence set forth in SEQ ID NO: 55. In anotherexample, the antibody or immunologically active fragment thereofcomprises a VH CDR1 sequence set forth in SEQ ID NO: 50, a VH CDR2sequence set forth in SEQ ID NO: 72, a VH CDR3 sequence set forth in SEQID NO: 52, a VL CDR1 set forth in SEQ ID NO: 53, a VL CDR2 sequence setforth in SEQ ID NO: 71, and a VL CDR3 sequence set forth in SEQ ID NO:55.

In one embodiment, the antibodies of the present invention bind to CD47in a head to side orientation that positions the heavy chain near themembrane of CD47 expressing cell, while the light chain occludes theSIRPα binding site on CD47. In another embodiment, the antibodies of thepresent invention bind to CD47 in a head to side orientation thatpositions the light chain near the membrane of CD47 expressing cell,while the heavy chain occludes the SIRPα binding site on CD47.

The CD47 antibodies bind to an epitope that includes any one of aminoacid residues 1-116 of CD47 when numbered in accordance with SEQ ID NO:147 (i.e., SEQ ID NO: 48 excluding the signal sequence (amino acids1-18)). For example, the antibodies of the present invention bind to anepitope that includes one or more of amino acid residues Q31, N32, T33,T34, E35, V36, Y37, V38, K39, W40, K41, F42, K43, G44, R45, D46, I47,Y48, T49, F50, D51, G52, A53, L54, N55, K56, S57, T58, V59, P60, T61,D62, F63, S64, S65, A66, K67, I68, E69, V70, S71, Q72, L73, L74, K75,G76, D77, A78, S79, L80, K81, M82, D83, K84, S85, D86, A87, V88, S89,H90, T91, G92, N93, Y94, T95, C96, E97, V98, T99, E100, L101, T102,R103, E104, G105, E106, T107, 1108, 1109, and E110 of CD47 when numberedin accordance with SEQ ID NO: 147.

In some cases, the antibodies of the present invention bind to adiscontinuous epitope that includes one or more of amino acid residuesY37, V38, K39, W40, K41, F42, K43, G44, R45, D46, I47, Y48, T49, F50,and D51 of CD47 when numbered in accordance with SEQ ID NO: 147. Forexample, the antibodies of the present invention bind to a discontinuousepitope comprising amino acids residues Y37, K39, K41, K43, G44, R45,D46, D51, H90, N93, E97, T99, E104, or E106 of CD47 when numbered inaccordance with SEQ ID NO: 147. For example, the antibodies of thepresent invention bind to a discontinuous epitope that includes at leastresidues of the KGRD (SEQ ID NO: 56) loop (residues 43-46) of CD47 whennumbered in accordance with SEQ ID NO: 147. For example, the antibodiesof the present invention bind to a discontinuous epitope that includesat least residues Y37, K39, K41, the KGRD (SEQ ID NO: 56) loop (residues43-46), D51, H90, N93, E97, T99, E104, and E106 of CD47 when numbered inaccordance with SEQ ID NO: 147. For example, the antibodies of thepresent invention bind to a discontinuous epitope that includes residuesY37, K39, K41, the KGRD (SEQ ID NO: 56) loop (residues 43-46), D51, H90,N93, E97, T99, E104, and E106 of CD47 when numbered in accordance withSEQ ID NO: 147.

The VH region of the CD47 antibodies described herein is primarilyinvolved in binding to the KGRD (SEQ ID NO: 56) loop of CD47. Thus, theunique epitope to which antibodies of the present invention bind is onthe side of CD47. In contrast to existing CD47 antibodies known in theart, the orientation of the VH domain of the CD47 antibodies describedherein in a membrane proximal position is a critical feature of theseantibodies that prevents cellular clumping, e.g., red blood cellhemagglutination, by constraining the antibodies such that they cannotbridge to CD47 molecules on adjacent cells. Additionally, because the VKdomain of the CD47 antibodies described herein interacts with apicalresidues such as Y37, T102, and E104, which are involved in SIRPαbinding, it is primarily the VK domain that physically precludes SIRPαbinding to CD47.

Also provided is an isolated antibody or an immunologically activefragment thereof which competes with the CD47 antibodies describedherein for preventing CD47 from interacting with SIRPα.

The invention provides a polypeptide comprising amino acids residuesY37, K39, K41, K43, G44, R45, D46, D51, H90, N93, E97, T99, E104, andE106 of CD47 when numbered in accordance with SEQ ID NO: 147. Alsoprovided is a polypeptide comprising any one of amino acid residues1-116 of CD47 when numbered in accordance with SEQ ID NO: 147. Forexample, the polypeptide comprises one or more of amino acid residuesQ31, N32, T33, T34, E35, V36, Y37, V38, K39, W40, K41, F42, K43, G44,R45, D46, I47, Y48, T49, F50, D51, G52, A53, L54, N55, K56, S57, T58,V59, P60, T61, D62, F63, S64, S65, A66, K67, 168, E69, V70, S71, Q72,L73, L74, K75, G76, D77, A78, S79, L80, K81, M82, D83, K84, S85, D86,A87, V88, S89, H90, T91, G92, N93, Y94, T95, C96, E97, V98, T99, E100,L101, T102, R103, E104, G105, E106, T107, 1108, 1109, and E110 of CD47when numbered in accordance with SEQ ID NO: 147. Also provided aremethods of using this polypeptide as an antigen, e.g., an antigen whichbinds a CD47 antibody.

The invention also provides methods of alleviating a symptom of a canceror other neoplastic condition by administering to a subject in needthereof one or more monoclonal antibodies that bind to CD47 or animmunologically active fragment thereof, wherein the antibody does notcause a significant level of hemagglutination of red blood cells, redblood cell depletion, anemia, and/or platelet depletion afteradministration. The antibody is administered in an amount sufficient toalleviate the symptom of the cancer or other neoplastic condition in thesubject. In some embodiments, the subject is a human. In someembodiments, the antibody is chimeric, humanized, or fully human. Insome embodiments, the antibody binds to human CD47. In some embodiments,the antibody or immunologically active fragment thereof prevents CD47from interacting with SIRPα. In some embodiments, the antibody orimmunologically active fragment thereof is an IgG isotype selected fromthe group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, andIgG4 isotype. In some embodiments, the antibody or immunologicallyactive fragment thereof is an IgG isotype selected from IgG4P andIgG4PE.

In some embodiments, the CD47 antibodies described herein are used inconjunction with one or more additional agents or a combination ofadditional agents. Suitable additional agents include currentpharmaceutical and/or surgical therapies for an intended application,such as, for example, cancer. For example, the CD47 antibodies can beused in conjunction with one or more additional chemotherapeutic oranti-neoplastic agents. Alternatively, the additional chemotherapeuticagent is radiotherapy. In some embodiments, the chemotherapeutic agentis a cell death-inducing agent. In some embodiments, thechemotherapeutic agent induces a loss of phospholipid asymmetry acrossthe plasma membrane, for example causes cell surface exposure ofphosphatidylserine (PS). In some embodiments, the chemotherapeutic agentinduces endoplasmic reticulum (ER) stress. In some embodiments, thechemotherapeutic agent is a proteasome inhibitor. In some embodiments,the chemotherapeutic agent induces the translocation of ER proteins tothe cell surface. In some embodiments, the chemotherapeutic agentinduces the translocation and cell surface exposure of calreticulin.

In some embodiments, the CD47 antibody and additional agent areformulated into a single therapeutic composition, and the CD47 antibodyand additional agent are administered simultaneously. Alternatively, theCD47 antibody and additional agent are separate from each other, e.g.,each is formulated into a separate therapeutic composition, and the CD47antibody and the additional agent are administered simultaneously, orthe CD47 antibody and the additional agent are administered at differenttimes during a treatment regimen. For example, the CD47 antibody isadministered prior to the administration of the additional agent, theCD47 antibody is administered subsequent to the administration of theadditional agent, or the CD47 antibody and the additional agent areadministered in an alternating fashion. As described herein, the CD47antibody and additional agent are administered in single doses or inmultiple doses.

One skilled in the art will appreciate that the antibodies of theinvention have a variety of uses. For example, the antibodies of theinvention are used as therapeutic agents, as reagents in diagnostic kitsor as diagnostic tools, or as reagents in competition assays to generatetherapeutic reagents.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this disclosure has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the disclosureencompassed by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph depicting the binding of CD47 on Daudi cells byantibodies in hybridoma supernatants as assessed by flow cytometry. FIG.1B is a graph showing the ability of some of the CD47 antibodies withinthe hybridoma supernatant to block the binding of recombinant humanSIRPα to recombinant human CD47, as determined by an ELISA.

FIGS. 2A-2B are a series of graphs depicting (FIG. 2A) the binding ofpurified murine CD47 antibodies to Raji cells, a cultured line oflymphoblastoid cells derived from a Burkitt lymphoma, and (FIG. 2B)CCRF-CEM cells, a CD47 positive human T cell lymphoblast-like cell line,as analyzed by flow cytometry. This experiment compares the binding ofthe murine antibodies of the present invention to the commerciallyavailable CD47 antibodies, B6H12 and 2D3.

FIGS. 3A-3B are a series of graphs depicting the capacity of CD47antibodies to block SIRPα binding using (FIG. 3A) an ELISA withrecombinant human protein, or (FIG. 3B) by flow cytometry using CCRF-CEMcells and recombinant human SIRPα protein.

FIGS. 4A-4F are a series of photographs, graphs, and a table that showRBC hemagglutination by CD47 antibodies. RBC hemagglutination isevidenced by a haze appearance in the well, whereas non-agglutinated RBCappear as punctate. FIG. 4A shows that the 2A1 antibody displays nohemagglutination at all concentrations tested. Hemagglutination index isdepicted in the graph. FIG. 4B shows that 2A1 is rare amongst many CD47antibodies in its inability to agglutinate RBCs. Also shown is the lackof agglutinating activity by the human chimeric version of 2A1 (2A1-xi).FIG. 4C shows that CD47 monoclonal antibody 2D3, which does not blockSIRPα, does not cause hemagglutination. FIG. 4D shows a highconcentration range of the CD47 antibodies in the hemagglutination assayand demonstrates the pro-zone effect. Hemagglutination index is depictedin the graph. FIG. 4E demonstrates the more narrow concentration rangefor hemagglutination by the CD47 antibody, 1B4, whereas this effect isabsent upon 2A1 binding. FIG. 4F shows that 2A1, chimeric 2A1 (2A1-xi),and humanized variants do not cause hemagglutination. In mostexperiments the 9E4 antibody and the commercial B6H12 antibody were usedas positive controls for hemagglutination. Other commercially availableantibodies used in these assays were the SIRPα blocking antibodies,BRC126 and CC2C6, and the non-SIRPα blocking antibody, 2D3.

FIG. 5 is a graph showing 2A1 and B6H12 binding to cynomolgus (cyno)monkey B-cells and Raji, as assessed by flow cytometry. 2A1 binds humanand cyno CD47 with equivalent affinity, as does B6H12, albeit with loweraffinity to both human and cyno CD47 than 2A1.

FIG. 6 is a graph depicting the binding of 2A1, 2A1-xi, and B6H12 toRaji cells, as assessed by flow cytometry. Importantly, the graph showsthat the variable region sequences of the heavy (VH) and light (VL)chains were correctly elucidated in the chimeric version of 2A1.

FIG. 7A-7J is a series of graphs showing binding of humanized variantsof 2A1 to Raji cells. 2A1-xi was used as an internal control on mostgraphs. Numerous combinations of heavy and light chains were tested asdescribed in Example 8.

FIG. 8A is image of the trace from size exclusion chromatography usingan AKTA FLPC with a Superdex200 column. Shown are the IgG1, IgG4P, andIgG4PE variants of the AB6.12 antibody. All three variant are over 97%monomeric. FIG. 8B is a photograph of a coomassie blue stained SDS-PAGEgel of numerous humanized variants of 2A1 under reducing (R) andnon-reducing (NR) conditions.

FIGS. 9A-9B are a series of graphs depicting the ability of CD47antibodies to promote phagocytosis of human tumor cell lines by humanmonocyte derived macrophages (MDM). FIG. 9A is a graph showing thephagocytic index, wherein antibodies used were the commercial antibodyB6H12, the murine 2A1 antibody, a humanized variant AB2.05 antibody, andthe non-blocking commercial antibody 2D3. FIG. 9B is a graph showing thephagocytic index, wherein the antibodies used were the commercialantibody B6H12, humanized antibody AB2.05 (human IgG1), and the IgG1,IgG4P, and IgG4PE variants of the humanized antibody AB6.12. CCRF-CEMcells were used as the CD47 target cell line in these experiments.

FIGS. 10A-10B are a series of graphs showing the anti-tumor effects ofCD47 antibodies in a Raji tumor model. FIG. 10A is a graph that showsthe efficacy of the murine antibodies 9E4, 1B4, 2A1, and the commercialantibody B6H12. FIG. 10B is a graph that shows the efficacy of the IgG1,IgG4P, and IgG4PE isotypes of the humanized antibody AB6.12, along withthe murine 2A1 antibody. In both models, mice were treated with 200 μgantibody doses three times a week.

FIGS. 11A-11C are a graphic representation of the co-crystal complexesof CD47-IgV with SIRPα-IgV domain (FIG. 11A, (Protein Data Bank (PDB)Reference No. 2JJS), B6H12 (FIG. 11B), and 2A1 (FIG. 11C). 2A1 and B6H12bind in very different orientations and distinct epitopes on CD47, bothof which overlap with the SIRPα binding site. The 2A1 antibody binds ina head to side orientation on the CD47 protein.

FIGS. 12A-12H are a series of graphs depicting the platelet levels inthe blood of cynomolgus monkeys following a single dose (vehicle, 10,30, or 100 mg/kg) of a CD47 antibody of IgG1, IgG4P (hinge stabilized:S228P), and IgG4PE (hinge stabilized: S228P, and further reduced FcγRbinding mutant: L235E) isotypes. The graphs in the left column (FIGS.12A, 12C, 12E, and 12G) present the mean platelet counts in whole bloodover time. The graphs in the right column (FIGS. 12B, 12D, 12F, and 12H)present the mean percent remaining platelets over time normalized to thepre-dose (4 days prior to injection) platelet count for each monkey.

FIG. 13 is a graph of the mean RBC counts from antibody-treatedcynomolgus monkeys, normalized to the mean RBC counts of the vehicletreated monkeys. The antibody-treated monkeys were administered variousdoses of the AB06.12-IgG4P or the AB06.12-IgG4PE antibodies of theinvention.

DETAILED DESCRIPTION

The present invention provides monoclonal antibodies that specificallybind CD47, including human CD47. These antibodies are collectivelyreferred to herein as CD47 antibodies.

The primary Fc dependent functions of an antibody for target cellelimination are complement dependent cytotoxicity (CDC) initiated bybinding C1q to the Fc region; antibody dependent cytotoxicity (ADCC)mediated by the interaction of the Fc region with Fcγ receptors (FcγRs),primary FcγRIIIa on immune effector cells (e.g., NK cells andNeutrophils); and antibody dependent cellular phagocytosis (ADCP) whichis carried out by macrophages through the recognition of opsinizedtarget cells via FcγRI. Antibody subclasses have differences in theirabilities to mediate Fc-dependent effector activities. In humans, theIgG1 and IgG3 subclasses have high potency for CDC due to binding C1q.In addition, the IgG1 subclass has the highest affinity for FcγRs and isthereby the most potent in terms of ADCC and Fc-dependent ADCP. The IgG4subclass is devoid of C1q binding ability and has greatly reduced FcγRbinding affinity and thereby has significantly diminished effectorfunction.

CD47, a multi-spanning transmembrane receptor belonging to theimmunoglobulin superfamily, interacts with SIRPα(signal-regulatory-protein a) on macrophages and thereby dampensphagocytosis. Cancer cells that co-opt this pathway evade phagocytosis.As described in detail below, this is a new mechanism of tumor immuneavoidance, and therapeutically targeting CD47 has widespread applicationin numerous cancers.

The expression of CD47 correlates with worse clinical outcomes in manydistinct malignancies including Non-Hodgkin Lymphoma (NHL), AcuteLymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), ovariancancer, glioma, glioblastoma, etc. In addition, CD47 has been identifiedas a cancer stem cell marker in both leukemias and solid tumors (Jaiswalet al., 2009 Cell, 138(2): 271-85; Chan et al., 2009 Proc Natl Acad SciUSA, 106(33): 14016-21; Chan et al., 2010 Curr Opin Urol, 20(5): 393-7;Majeti R et al., 2011 Oncogene, 30(9): 1009-19).

CD47 blocking antibodies have demonstrated anti-tumor activity inmultiple in vivo tumor models. Furthermore, these antibodies have beenshown to synergize with other therapeutic antibodies including Rituxan®and Herceptin® in tumor models. Blocking the interaction of CD47 withSIRPα is capable of promoting phagocytosis of CD47 expressing cells bymacrophages (reviewed in Chao et al., 2012 Curr Opin Immunol, 24(2):225-32). Mice lacking CD47 are markedly resistant to radiation therapy,suggesting a role for targeting CD47 in combination with radiotherapy(Isenberg et al., 2008 Am J Pathol, 173(4): 1100-1112; Maxhimer et al.,2009 Sci Transl Med, 1(3): 3ra7). Furthermore, syngeneic tumor models inthese mice display decreased bone metastasis compared wild-type mice(Uluckan et al., 2009 Cancer Res, 69(7): 3196-204).

Importantly, most CD47 antibodies have been reported to causehemagglutination of human erythrocytes as well as red blood celldepletion and anemia. Hemagglutination is an example of a homotypicinteraction, wherein two CD47 expressing cells are caused to aggregateor clump when treated with a bivalent CD47 binding entity. For example,the CD47 antibody, MABL, as a full IgG or F(ab′)2, has been reported tocause hemagglutination of erythrocytes, and, only when MABL was alteredinto an scFv or bivalent scFv, was this effect mitigated. (See e.g., UnoS, Kinoshita Y, Azuma Y et al. Antitumor activity of a monoclonalantibody against CD47 in xenograft models of human leukemia. Oncol Rep2007; 17: 1189-94; Kikuchi Y, Uno S, Yoshimura Y et al. A bivalentsingle-chain Fv fragment against CD47 induces apoptosis for leukemiccells. Biochem Biophys Res Commun 2004; 315: 912-8). Other known CD47antibodies including B6H12, BRC126, and CC2C6 also causehemagglutination of RBCs, as described in detail below.

In addition, CD47 antibodies and CD47 antagonizing SIRPα-Fc fusionproteins have been reported to cause red blood cell depletion and anemiawhen administered to mice and/or cynomolgus monkeys. (See, Weiskopf etal. Engineered SIRPa Variants as Immunotherapeutic Adjuvants toAnticancer Antibodies. Science 2013; 341:88). Anemia is a condition inwhich the blood lacks a sufficient amount of red blood cells orhemoglobin to carry oxygen to the tissues. Anemia can be diagnosed by anumber of methods generally known in the art. For example, anemia isdiagnosed by determining the complete blood count (CBC), whichdetermines the number, size, volume, and hemoglobin content of red bloodcells. Anemia is also diagnosed by measuring the blood iron level and/orserum ferritin level, which are indicators of the body's total ironstores. In addition, anemia is diagnosed by measuring the levels ofvitamin B12 and folate, reticulocyte count, and bilirubin.

Thus, the aggregation of cells, RBC depletion, and anemia representmajor limitations of therapeutically targeting CD47 with existing fullIgG antibodies and/or SIRPα-Fc fusion proteins.

Moreover, an important characteristic of CD47 antibodies is the abilityto block the interaction of CD47 and SIRPα in order to promote thephagocytosis of CD47 expressing cells by macrophages. Many existing CD47antibodies block SIRPα; however, prior to the invention describedherein, existing antibodies that blocked SIRPα caused the side effect ofhemagglutination, which, as described above, is undesirable. Otherexisting antibodies, such as 2D3, do not cause hemagglutination;however, these antibodies also do not block SIRPα, rendering themineffective in the promotion of phagocytosis. Thus, prior to theinvention described herein, there was a pressing need to identify CD47antibodies that blocked SIRPα without causing cellular clumping.

The CD47 antibodies of the present invention avoid the undesirableeffect of hemagglutination, thereby increasing the efficacy oftherapeutically targeting CD47, and maintain the ability to block theinteraction of CD47 with SIRPα, thereby promoting phagocytosis of CD47expressing cells. Specifically, the full IgG CD47 antibodies of thepresent invention (e.g., 2A1 and its humanized derivatives includingthose provided in Table 1) do not agglutinate cells at a significantlevel. For example, the CD47 antibodies of the invention do nothemagglutinate RBCs at a significant level. Described herein are thefirst CD47 antibodies in a full IgG format that block SIRPα and do notcause a significant level of hemagglutination and/or RBC depletion.Taken together, the antibodies of the invention (e.g., the 2A1 antibodyand its humanized derivatives) are unique among existing CD47 antibodiesin their ability to block SIRPα, but not cause a significant level ofhemagglutination and/or RBC depletion.

The CD47 antibodies of the invention exhibit numerous desirablecharacteristics, such as, by way of non-limiting example, potentblocking of the interaction between CD47 and its ligand SIRPα, withoutcausing a significant level of or otherwise modulating hemagglutinationof erythrocytes, as well as potent anti-tumor activity. For example, theCD47 antibodies of the invention block at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 95%, or at least 99% ofthe interaction between CD47 and SIRPα as compared to the level ofinteraction between CD47 and SIRPα in the absence of the CD47 antibodydescribed herein. The CD47 antibodies of the invention do not cause asignificant level of agglutination of cells, e.g., the CD47 antibodiesof the invention do not cause a significant level of hemagglutination ofred blood cells. For example, the level of agglutination in the presenceof the CD47 antibodies of the invention is reduced by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 99%compared to the level of agglutination in the presence existing CD47antibodies. In some embodiments, the CD47 antibodies of the invention donot cause a significant level of agglutination if the level ofagglutination in the presence of the CD47 antibodies of the invention isreduced by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 99% compared to the level of agglutination in thepresence of CD47 antibody, 1B4, which comprises a variable heavy andvariable light chain sequence provided in SEQ ID NO: 80 and SEQ ID NO:81, respectively. The CD47 antibodies of the invention do not cause asignificant level of RBC depletion. For example, the RBC count in asubject after administration (5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h,5 h, 12 h, 24 h, 2 days, 4 days, 6 days, 1 week, 2 weeks, 3 weeks, 1month, 2 months, or more) of an antibody of the invention is at least50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5% of the RBC countprior to administration. Alternatively or in addition, the RBC count ina subject after administration (5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4h, 5 h, 12 h, 24 h, 2 days, 4 days, 6 days, 1 week, 2 weeks, 3 weeks, 1month, 2 months, or more) of an antibody of the invention is at least50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5% of the RBC count in asubject after administration of a placebo treatment (e.g., vehicle). RBCcounts are determined by standard methods in the art. The antibodies ofthe present invention are also significantly more potent in tumor modelscompared to antibodies known in the art. For example, the ability ofmacrophages to phagocytose tumor cells in the presence of CD47antibodies of the invention is increased by at least 5%, at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 99% compared to theability of macrophages to phagocytose tumor cells in the presence ofexisting CD47 antibodies.

Those skilled in the art will recognize that it is possible toquantitate, without undue experimentation, the level of agglutination,e.g., the level of hemagglutination of RBCs. For example, those skilledin the art will recognize that the level of hemagglutination isascertained by measuring the area of an RBC dot after performing ahemagglutination assay in the presence of the CD47 antibodies of theinvention, as described in the Examples below. In some cases, the areaof the RBC dot in the presence of the CD47 antibody of the invention iscompared to the area of the RBC dot in the absence of a CD47 antibody,i.e., in the presence of zero hemagglutination. In this manner,hemagglutination is quantified relative to a baseline control. A largerRBC dot area corresponds to a higher level of hemagglutination.Alternatively, densitometry of the RBC dot may also be utilized toquantitate hemagglutination.

In addition, antibodies, such as CD47 antibodies of the invention, canplay a role in platelet depletion (e.g., in a Fc-dependent manner) uponadministration. For example, treatment of a cynomolgus monkey with anantibody of the IgG1 subclass that binds to CD47 can result insignificant depletion of platelets at multiple doses. See, e.g., Example12 and FIG. 12C-D. A disadvantage of platelet depletion is that, whensevere, it can result in fatal hemorrahaging. The present invention isbased in part on the surprising discovery that mutation of an antibodyto diminish FcγR binding results in undetectable to low levels ofplatelet depletion even at high doses (e.g., 100 mg/kg). See, e.g.,Example 12 and FIG. 12G-H. Thus, a CD47 binding antibody with severelyreduced FcγR binding and effector function does not result in plateletdepletion.

Platelet counts can be measured using routine methods generally known toone of skill in the art. Remaining platelet percentage over time can becalculated as the platelet count remaining at a certain time point afteradministration of a therapy of the invention (e.g., a CD47 antibody)normalized to the platelet count sometime before (e.g., 1 hour, 3 hours,6 hours, 12 hours, 1 day, 2, days, 4 days, 5, days, 6, days, or more)administration of the therapy. Significant platelet depletion can bedefined as a remaining platelet percentage after administration of lessthan 100% (e.g., less than 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%,30%, 20%, or 10%). The therapies of the present invention (e.g.,antibodies) lead to an insignificant level of platelet depletion (e.g.,a remaining platelet percentage after administration of at least 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100%). The CD47 antibodies of theinvention bind to human CD47 and block its interaction with SIRPα (FIGS.1B, 3, and 7J). These antibodies do not cause a significant level ofhemagglutination of human erythrocytes (FIG. 4). Also, these antibodiescan possess the property of not causing significant levels of plateletdepletion (e.g., Example 12 and FIG. 12). These antibodies are capableof promoting phagocytosis of tumor cells by macrophages (FIG. 9).Furthermore, the CD47 antibodies display potent anti-tumor activity in amouse model of human lymphoma (FIG. 10). Thus, the CD47 antibodies ofthe invention circumvent a major limiting factor for the therapeutictargeting CD47. Accordingly, the CD47 antibodies of the invention standto be of great importance in treatment a multitude of cancers.

Antibodies of the invention that specifically bind human CD47, block,inhibit, disrupt or otherwise modulate the interaction between humanCD47 and human SIRPα, without causing a significant level of orotherwise modulating hemagglutination of erythrocytes.

The antibodies of the present invention bind to a CD47 epitope with anequilibrium binding constant (K_(d)) of ≦1 μM, e.g., 100 nM, preferably10 nM, and more preferably ≦1 nM. For example, the CD47 antibodiesprovided herein exhibit a K_(d) in the range approximately between ≦1 nMto about 1 pM.

The CD47 antibodies of the invention serve to modulate, block, inhibit,reduce, antagonize, neutralize or otherwise interfere with thefunctional activity of the widely distributed CD47. Functionalactivities of CD47 include for example, signaling via the interactionwith SIRPα, modulating, e.g., increasing, intracellular calciumconcentration upon cell adhesion to extracellular matrix, interactingwith the C-terminal cell binding domain of thrombospondin, interactingwith fibrinogen, and interacting with various integrins. For example,the CD47 antibodies completely or partially inhibit CD47 functionalactivity by partially or completely modulating, blocking, inhibiting,reducing antagonizing, neutralizing, or otherwise interfering with thebinding of CD47 to SIRPα.

The CD47 antibodies are considered to completely modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere with CD47functional activity when the level of CD47 functional activity in thepresence of CD47 antibody is decreased by at least 95%, e.g., by 96%,97%, 98%, 99% or 100% as compared to the level of CD47 functionalactivity in the absence of binding with a CD47 antibody describedherein. The CD47 antibodies are considered to significantly block,inhibit, reduce, antagonize, neutralize or otherwise interfere with CD47functional activity when the level of CD47 activity in the presence ofthe CD47 antibody is decreased by at least 50%, e.g., 55%, 60%, 75%,80%, 85% or 90% as compared to the level of CD47 activity in the absenceof binding with a CD47 antibody described herein. The CD47 antibodiesare considered to partially modulate, block, inhibit, reduce,antagonize, neutralize or otherwise interfere with CD47 functionalactivity when the level of CD47 activity in the presence of the CD47antibody is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%,50%, 60%, 75%, 80%, 85% or 90% as compared to the level of CD47 activityin the absence of binding with a CD47 antibody described herein.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms CD47, integrin-associated protein (IAP),ovarian cancer antigen OA3, Rh-related antigen and MER6 are synonymousand may be used interchangeably.

The terms red blood cell(s) and erythrocyte(s) are synonymous and usedinterchangeably herein.

The term agglutination refers to cellular clumping, while the termhemagglutination refers to clumping of a specific subset of cells, i.e.,red blood cells. Thus, hemagglutination is a type of agglutination.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” “or directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at much loweraffinity (K_(d)>10-6). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,Fab, Fab′ and F(ab′)2 fragments, Fv, scFvs, and an Fab expressionlibrary.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses (also known as isotypes) as well, such as IgG₁, IgG₂, andothers. Furthermore, in humans, the light chain may be a kappa chain ora lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three-dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or fragment thereof, ora T-cell receptor. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 μM;e.g., ≦100 nM, preferably ≦10 nM and more preferably ≦1 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (k_(on)) andthe “off rate constant” (k_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of k_(off)/k_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to CD47, when the equilibrium binding constant(K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably ≦10 nM, and mostpreferably ≦100 pM to about 1 pM, as measured by assays such asradioligand binding assays, surface plasmon resonance (SPR), flowcytometry binding assay, or similar assays known to those skilled in theart.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide,” as referred to herein, refers to apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long′ morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto CD47, under suitable binding conditions. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, preferably at least 50 aminoacids long or longer, and can often be as long as a full-lengthnaturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med.Chem. 30:1229 (1987). Such compounds are often developed with the aid ofcomputerized molecular modeling. Peptide mimetics that are structurallysimilar to therapeutically useful peptides may be used to produce anequivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ₃H, ₁₄C, ₁₅N, ₃₅S, ₉₀Y, ₉₉Tc,₁₁₁In, ₁₂₅I, ₁₃₁I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

CD47 Antibodies

Monoclonal antibodies of the invention have the ability to bind CD47, toinhibit the binding of SIRPα to CD47, decrease CD47-SIRPα-mediatedsignaling, promote phagocytosis, and to inhibit tumor growth and/ormigration. Inhibition is determined, for example, using the cellularassay described herein in the Examples.

Exemplary antibodies of the invention include the 2A1 antibody, thechimeric version of 2A1, and humanized variants of 2A1. Exemplaryantibodies of the invention include an antibody having a variable heavy(VH) chain selected from SEQ ID NOs: 5-30, and having a variable light(VL) chain selected from SEQ ID NOs: 31-47. Specifically, exemplaryantibodies include those provided in Table 1.

TABLE 1 Antibody Variable heavy (VH) chain Variable light (VL) chain 2A1SEQ ID NO: 5 SEQ ID NO: 31 2A1-xi SEQ ID NO: 5 SEQ ID NO: 32 AB2.03 SEQID NO: 7 SEQ ID NO: 33 AB2.04 SEQ ID NO: 7 SEQ ID NO: 34 AB2.05 SEQ IDNO: 7 SEQ ID NO: 35 AB2.06 SEQ ID NO: 7 SEQ ID NO: 36 AB2.07 SEQ ID NO:7 SEQ ID NO: 37 AB2.08 SEQ ID NO: 7 SEQ ID NO: 38 AB2.09 SEQ ID NO: 7SEQ ID NO: 39 AB2.13 SEQ ID NO: 7 SEQ ID NO: 43 AB3.09 SEQ ID NO: 8 SEQID NO: 39 AB6.12 SEQ ID NO: 11 SEQ ID NO: 42 AB6.13 SEQ ID NO: 11 SEQ IDNO: 43 AB6.14 SEQ ID NO: 11 SEQ ID NO: 44 AB6.17 SEQ ID NO: 11 SEQ IDNO: 47 AB10.13 SEQ ID NO: 15 SEQ ID NO: 43 AB10.14 SEQ ID NO: 15 SEQ IDNO: 44 AB11.05 SEQ ID NO: 16 SEQ ID NO: 35 AB12.05 SEQ ID NO: 17 SEQ IDNO: 35 AB15.05 SEQ ID NO: 20 SEQ ID NO: 35 AB16.05 SEQ ID NO: 21 SEQ IDNO: 35 AB17.05 SEQ ID NO: 22 SEQ ID NO: 35 AB22.05 SEQ ID NO: 27 SEQ IDNO: 35 AB23.05 SEQ ID NO: 28 SEQ ID NO: 35 AB24.05 SEQ ID NO: 29 SEQ IDNO: 35 AB25.05 SEQ ID NO: 30 SEQ ID NO: 35

Also included in the invention are antibodies that bind to the sameepitope as the CD47 antibodies described herein. For example, antibodiesof the invention specifically bind to an epitope that includes one ormore amino acid residues on human CD47 (see e.g., GenBank Accession No.Q08722.1).

The amino acid sequence of an exemplary human CD47 is provided below(GenBank Accession No. Q08722.1 (GI:1171879), incorporated herein byreference). The signal sequence (amino acids 1-18) is underlined.

(SEQ ID NO: 48)   1mwplvaalll gsaccgsaql lfnktksvef tfcndtvvip cfvtnmeaqn ttevyvkwkf  61kgrdiytfdg alnkstvptd fssakievsq llkgdaslkm dksdavshtg nytcevtelt 121regetiielk yrvvswfspn enilivifpi faillfwgqf giktlkyrsg gmdektiall 181vaglvitviv ivgailfvpg eyslknatgl glivtstgil illhyyvfst aigltsfvia 241ilviqviayi lavvglslci aacipmhgpl lisglsilal aqllglvymk fvasnqktiq 301pprkaveepl nafkeskgmm nde

For clarity, the amino acid sequence of an exemplary human CD47excluding the signal sequence is provided below.

(SEQ ID NO: 147)   1qllfnktksv eftfcndtvv ipcfvtnmea qnttevyvkw kfkgrdiytf dgalnkstvp  61tdfssakiev sqllkgdasl kmdksdavsh tgnytcevte ltregetiie lkyrvvswfs 121pnenilivif pifaillfwg qfgiktlkyr sggmdektia llvaglvitv ivivgailfv 181pgeyslknat glglivtstg ilillhyyvf staigltsfv iailviqvia yilavvglsl 241ciaacipmhg pilisglsil alaqllglvy mkfvasnqkt iqpprkavee plnafkeskg 301mmnde 

The amino acid sequence of an exemplary human CD47-IgV domain isprovided below:

(SEQ ID NO: 49) 19qllfnktksv eftfcndtvv ipcfvtnmea qnttevyvkw kfkgrdiytf dgalnkstvp 79tdfssakiev sqllkgdasl kmdksdaysh tgnytcevte ltregetiie lkyrvv

Exemplary monoclonal antibodies of the invention include, for example,humanized antibodies having a variable heavy chain region (VH) and/orvariable light (VL) chain region shown in the sequences below.

Variable heavy (VH) chain regions of the CD47 antibodies are providedbelow. The complementarity determining regions (CDRs) of the VH chain ofthe CD47 antibodies are highlighted below. In some embodiments, theamino acid sequence of VH CDR1 is GFNIKDYYLH (SEQ ID NO: 50), GYTFTYYYLH(SEQ ID NO: 57), GFTFTYYYLH (SEQ ID NO: 58), GYNFTYYYLH (SEQ ID NO: 59),GYTITYYYLH (SEQ ID NO: 60), GYTFKYYYLH (SEQ ID NO: 61), GYTFTDYYLH (SEQID NO: 62), GFTFTDYYLH (SEQ ID NO: 63), GFTITDYYLH (SEQ ID NO: 64),GYTFKDYYLH (SEQ ID NO: 65), or GFTFKDYYLH (SEQ ID NO: 66). In someembodiments, the amino acid sequence of VH CDR2 is WIDPDNGDTE (SEQ IDNO: 51), WIDPDQGDTE (SEQ ID NO: 72), WIDPDYGDTE (SEQ ID NO: 73),WIDPDSGDTE (SEQ ID NO: 74), WIDPDNADTE (SEQ ID NO: 75), or WIDPDNTDTE(SEQ ID NO: 76). In some embodiments, the amino acid sequence of VH CDR3is NAAYGSSSYPMDY (SEQ ID NO: 52) or NAAYGSSPYPMDY (SEQ ID NO: 77).

(SEQ ID NO: 5) EVQLQQSGAELVRSGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPDNGDTEFAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAAY GSSSYPMDYWGQGTSVTV (SEQ ID NO: 6) EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVQQAPGKGLEWMGWIDPDNGDTEYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 7) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 8) EVQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 9) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQGRVTMTADTSSNTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 10) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 11) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDQGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 12) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDYGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 13) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDSGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 14) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNADTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 15) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNTDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 16) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSPYPMDYWGQGTTVTV (SEQ ID NO: 17) QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 18) QMQLVQSGAEVKKTGSSVKVSCKASGFTFTYYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 19) QMQLVQSGAEVKKTGSSVKVSCKASGYNFTYYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 20) QMQLVQSGAEVKKTGSSVKVSCKASGYTITYYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 21) QMQLVQSGAEVKKTGSSVKVSCKASGYTFKYYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 22) QMQLVQSGAEVKKTGSSVKVSCKASGYTFTDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 23) QMQLVQSGAEVKKTGSSVKVSCKASGFTFTDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 24) QMQLVQSGAEVKKTGSSVKVSCKASGFTITDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 25) QMQLVQSGAEVKKTGSSVKVSCKASGYTFKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 26) QMQLVQSGAEVKKTGSSVKVSCKASGFTFKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 27) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYLQLSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 28) QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLTSEDTAVYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 29) EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVRQAPGQALEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV (SEQ ID NO: 30) EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVQQAPGKGLEWMGWIDPDNGDTEYAQKFQDRVTITRDRSMSTAYMELSSLRSEDTAMYYCNAAY GSSSYPMDYWGQGTTVTV 

Variable light (VL) chain regions of the CD47 antibodies are providedbelow. The CDRs of the VL chain of the CD47 antibodies are highlightedbelow. In some embodiments, the amino acid sequence of VL CDR1 isKASQDIHRYLS (SEQ ID NO: 53), RASQDIHRYLA (SEQ ID NO: 67), or RARQGIHRYLS(SEQ ID NO: 68). In some embodiments, the amino acid sequence of VL CDR2is RANRLVD (SEQ ID NO: 54), RANRLQS (SEQ ID NO: 69), RANRRAT (SEQ ID NO:70), or RANRLVS (SEQ ID NO: 71). In some embodiments, the amino acidsequence of VL CDR3 is LQYDEFPYT (SEQ ID NO: 55).

(SEQ ID NO: 31) DIKMTQSPSSLYASLGERVTITCKASQDIHRYLSWFQQKPGKSPKILIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGG GTKLEMK(SEQ ID NO: 32) DIKMTQSPSSLYASLGERVTITCKASQDIHRYLSWFQQKPGKSPKILIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGG GTKLEIK(SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCKASQDIHRYLSWYQQKPGKAPKLLIYRANRLVDGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPYTFGG GTKVEIK(SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITCKASQDIHRYLSWFQQKPGKAPKSLIYRANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 35) NIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKHLIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 36) DIQMTQSPSSLSASVGDRVTITCKASQDIHRYLSWYQQKPGKAPKRLIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 37) DIQMTQSPSSLSASVGDRVTITCRASQDIHRYLAWYQQKPGKVPKLLIYRANRLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLQYDEFPYTFGQ GTKVEIK (SEQ ID NO: 38) EIVLTQSPATLSLSPGERATLSCRASQDIHRYLAWYQQKPGQAPRLLIYRANRRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQYDEFPYTGFQ GTRLEIK (SEQ ID NO: 39) DIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKHLIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 40) NIQMTQSPSAMSASVGDRVTITCRARQGIHRYLSWFQQKPGKVPKHLIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 41) NIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKILIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 42) NIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKHLIYRANRLVSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 43) NIQMTQSPSAMSASVGDRVTITCRARQGIHRYLSWFQQKPGKVPKILIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 44) NIQMTQSPSAMSASVGDRVTITCRARQGIHRYLSWFQQKPGKVPKHLIYRANRLVSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 45) NIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKLLIYRANRLVDGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 46) NIQMTQSPSAMSASVGDRVTITCKASQDIHRYLSWFQQKPGKVPKLLIYRANRLVSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK (SEQ ID NO: 47) NIQMTQSPSAMSASVGDRVTITCRARQGIHRYLSWFQQKPGKVPKLLIYRANRLVSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK 

In some cases, the CD47 antibodies described herein comprise a variableheavy chain region selected from SEQ ID NOs: 5-30 and a variable lightchain region selected from SEQ ID NOs: 31-47. An exemplary CD47 antibodycomprises a variable heavy chain region set forth in SEQ ID NO: 5 and avariable light chain region set forth in SEQ ID NO: 31; a variable heavychain region set forth in SEQ ID NO: 7 and a variable light chain regionset forth in SEQ ID NO: 35; a variable heavy chain region set forth inSEQ ID NO: 11 and a variable light chain region set forth in SEQ ID NO:42, a variable heavy chain region set forth in SEQ ID NO: 5 and avariable light chain region set forth in SEQ ID NO: 32, a variable heavychain region set forth in SEQ ID NO: 7 and a variable light chain regionset forth in SEQ ID NO: 33, a variable heavy chain region set forth inSEQ ID NO: 7 and a variable light chain region set forth in SEQ ID NO:34, a variable heavy chain region set forth in SEQ ID NO: 7 and avariable light chain region set forth in SEQ ID NO: 36, a variable heavychain region set forth in SEQ ID NO: 7 and a variable light chain regionset forth in SEQ ID NO: 37, a variable heavy chain region set forth inSEQ ID NO: 7 and a variable light chain region set forth in SEQ ID NO:38, a variable heavy chain region set forth in SEQ ID NO: 29 and avariable light chain region set forth in SEQ ID NO: 35, a variable heavychain region set forth in SEQ ID NO: 30 and a variable light chainregion set forth in SEQ ID NO: 35, a variable heavy chain region setforth in SEQ ID NO: 7 and a variable light chain region set forth in SEQID NO: 43, a variable heavy chain region set forth in SEQ ID NO: 11 anda variable light chain region set forth in SEQ ID NO: 43, a variableheavy chain region set forth in SEQ ID NO: 11 and a variable light chainregion set forth in SEQ ID NO: 47, a variable heavy chain region setforth in SEQ ID NO: 15 and a variable light chain region set forth inSEQ ID NO: 43, a variable heavy chain region set forth in SEQ ID NO: 15and a variable light chain region set forth in SEQ ID NO: 44, a variableheavy chain region set forth in SEQ ID NO: 11 and a variable light chainregion set forth in SEQ ID NO: 44, a variable heavy chain region setforth in SEQ ID NO: 22 and a variable light chain region set forth inSEQ ID NO: 35, a variable heavy chain region set forth in SEQ ID NO: 7and a variable light chain region set forth in SEQ ID NO: 39, a variableheavy chain region set forth in SEQ ID NO: 8 and a variable light chainregion set forth in SEQ ID NO: 39, a variable heavy chain region setforth in SEQ ID NO: 16 and a variable light chain region set forth inSEQ ID NO: 35, a variable heavy chain region set forth in SEQ ID NO: 20and a variable light chain region set forth in SEQ ID NO: 35, a variableheavy chain region set forth in SEQ ID NO: 21 and a variable light chainregion set forth in SEQ ID NO: 35, a variable heavy chain region setforth in SEQ ID NO: 17 and a variable light chain region set forth inSEQ ID NO: 35, a variable heavy chain region set forth in SEQ ID NO: 28and a variable light chain region set forth in SEQ ID NO: 35, or avariable heavy chain region set forth in SEQ ID NO: 27 and a variablelight chain region set forth in SEQ ID NO: 35.

The CD47 antibodies described herein comprise any one of the VH regionsprovided in SEQ ID NOs: 5-30 paired with any one of the VL regionsprovided in SEQ ID NOs: 31-47. Specifically, the CD47 antibodiesdescribed herein comprise any one of the VH regions provided in SEQ IDNOs: 5, 7, 8, 11, 15-17, 20-22, and 27-30 paired with any one of the VLregions provided in SEQ ID NOs: 31-39, 42, 43, 44, and 47.

The CD47 antibodies described herein comprise any one of the VH CDR1regions provided in SEQ ID NO: 50, SEQ ID NO: 57, SEQ ID NO: 58, SEQ IDNO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, any one of the VH CDR2regions provided in SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 73, SEQ IDNO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, any one of the VH CDR3 regionsprovided in SEQ ID NO: 52 and SEQ ID NO: 77, any one of the VL CDR1regions provided in SEQ ID NO: 53, SEQ ID NO: 67, and SEQ ID NO: 68, anyone of the VL CDR2 regions provided in SEQ ID NO: 54, SEQ ID NO: 69, SEQID NO: 70, and SEQ ID NO: 71, and the VL CDR3 region provided in SEQ IDNO: 55.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody hasthe same specificity as a monoclonal antibody of the invention (e.g.,the 2A1 antibody, or an antibody having a variable heavy chain selectedfrom SEQ ID NOs: 5-31, and a variable light chain selected from SEQ IDNOs: 31-47) by ascertaining whether the former prevents the latter frombinding to CD47. If the monoclonal antibody being tested competes withthe monoclonal antibody of the invention, as shown by a decrease inbinding by the monoclonal antibody of the invention, then the twomonoclonal antibodies bind to the same, or a closely related, epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with soluble CD47protein (with which it is normally reactive), and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind CD47. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

Antibodies of the Present Invention

Screening of monoclonal antibodies of the invention, can be also carriedout, e.g., by measuring CD47- and/or CD47/SIRPα-mediated signaling, anddetermining whether the test monoclonal antibody is able to modulate,block, inhibit, reduce, antagonize, neutralize or otherwise interferewith CD47- and/or CD47/SIRPα-mediated signaling. These assays caninclude competitive binding assays. Additionally, these assays canmeasure a biologic readout, for example the ability to promotephagocytosis of a CD47 expressing cell by a macrophage, as is describedin Example 9 (FIG. 9).

Various procedures known within the art may be used for the productionof monoclonal antibodies directed against CD47, or against derivatives,fragments, analogs homologs or orthologs thereof. (See, for example,Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated hereinby reference). Fully human antibodies are antibody molecules in whichthe entire sequence of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies” or “fully human antibodies” herein. Human monoclonalantibodies are prepared, for example, using the procedures described inthe Examples provided below. Human monoclonal antibodies can be alsoprepared by using the trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may beproduced by using human hybridomas (see Cote, et al., 1983. Proc NatlAcad Sci USA 80: 2026-2030) or by transforming human B-cells withEpstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The CD47 antibodies of the invention are monoclonal antibodies.Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with CD47- and/or CD47/SIRPα-mediatedcell signaling are generated, e.g., by immunizing an animal withmembrane bound and/or soluble CD47, such as, for example, human CD47 oran immunogenic fragment, derivative or variant thereof. Alternatively,the animal is immunized with cells transfected with a vector containinga nucleic acid molecule encoding CD47 such that CD47 is expressed andassociated with the surface of the transfected cells. Alternatively, theantibodies are obtained by screening a library that contains antibody orantigen binding domain sequences for binding to CD47. This library isprepared, e.g., in bacteriophage as protein or peptide fusions to abacteriophage coat protein that is expressed on the surface of assembledphage particles and the encoding DNA sequences contained within thephage particles (i.e., “phage displayed library”). Hybridomas resultingfrom myeloma/B cell fusions are then screened for reactivity to CD47.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of monoclonalantibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as Chinese hamster ovary (CHO) cells, HumanEmbryonic Kidney (HEK) 293 cells, simian COS cells, PER.C6®, NS0 cells,SP2/0, YB2/0, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also can be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences (seeU.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Human Antibodies and Humanization of Antibodies

Monoclonal antibodies of the invention include fully human antibodies orhumanized antibodies. These antibodies are suitable for administrationto humans without engendering an immune response by the human againstthe administered immunoglobulin.

A CD47 antibody is generated, for example, using the proceduresdescribed in the Examples provided below. For example, CD47 antibodiesof the invention are identified using a modified RIMMS (RepetitiveImmunization Multiple Sites) immunization strategy in mice andsubsequent hybridoma generation.

In other, alternative methods, a CD47 antibody is developed, forexample, using phage-display methods using antibodies containing onlyhuman sequences. Such approaches are well-known in the art, e.g., inWO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated byreference. In this approach, a combinatorial library of phage carryingrandom pairs of light and heavy chains are screened using natural orrecombinant source of cd47 or fragments thereof. In another approach, aCD47 antibody can be produced by a process wherein at least one step ofthe process includes immunizing a transgenic, non-human animal withhuman CD47 protein. In this approach, some of the endogenous heavyand/or kappa light chain loci of this xenogenic non-human animal havebeen disabled and are incapable of the rearrangement required togenerate genes encoding immunoglobulins in response to an antigen. Inaddition, at least one human heavy chain locus and at least one humanlight chain locus have been stably transfected into the animal. Thus, inresponse to an administered antigen, the human loci rearrange to providegenes encoding human variable regions immunospecific for the antigen.Upon immunization, therefore, the xenomouse produces B-cells thatsecrete fully human immunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181and U.S. Pat. No. 6,150,584, which is hereby incorporated by referencein its entirety. This general strategy was demonstrated in connectionwith generation of the first XenoMouse™ strains as published in 1994.See Green et al. Nature Genetics 7:13-21 (1994), which is herebyincorporated by reference in its entirety. See also, U.S. Pat. Nos.6,162,963; 6,150,584; 6,114,598; 6,075,181; and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and EuropeanPatent No., EP 0 463 151 B1 and International Patent Applications No. WO94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related familymembers.

In an alternative approach, others have utilized a “minilocus” approachin which an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more VH genes,one or more D_(H) genes, one or more J_(H) genes, a mu constant region,and a second constant region (preferably a gamma constant region) areformed into a construct for insertion into an animal. See e.g., U.S.Pat. Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825;5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429;5,789,215; 5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and6,255,458; and European Patent No. 0 546 073 B1; and InternationalPatent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO97/13852, and WO 98/24884 and related family members.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and an immune variable region,it is expected that certain human anti-chimeric antibody (HACA)responses will be observed, particularly in chronic or multi-doseutilizations of the antibody. Thus, it would be desirable to providefully human antibodies against CD47 in order to vitiate or otherwisemitigate concerns and/or effects of HAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92102190 and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,761; 5,693,792; 5,714,350; and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,IgG2, IgG3, and IgG4. Either of the human light chain constant regions,kappa or lambda, may be used. The chimeric, humanized antibody is thenexpressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g., SV-40 earlypromoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as willbe appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPlückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743. If display technologies areutilized to produce antibodies that are not human, such antibodies canbe humanized as described above.

Using these techniques, antibodies can be generated to CD47 expressingcells, soluble forms of CD47, epitopes or peptides thereof, andexpression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed herein.

The CD47 antibodies of the invention can be expressed by a vectorcontaining a DNA segment encoding the single chain antibody describedabove.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G., et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofCD47 in a sample. The antibody can also be used to try to bind to anddisrupt CD47- and/or the CD47/SIRPα interaction and CD47/SIRPα-mediatedsignaling.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fabfragments with the desired specificity for a protein or derivatives,fragments, analogs or homologs thereof. Antibody fragments that containthe idiotypes to a protein antigen may be produced by techniques knownin the art including, but not limited to: (i) an F(ab′)2 fragmentproduced by pepsin digestion of an antibody molecule; (ii) an Fabfragment generated by reducing the disulfide bridges of an F_((ab′)2)fragment; (iii) an Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) F_(v)fragments.

The invention also includes Fv, Fab, Fab′ and F(ab′)2 CD47 fragments,single chain CD47 antibodies, single domain antibodies (e.g., nanobodiesor VHHs), bispecific CD47 antibodies, and heteroconjugate CD47antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for CD47. The second binding target is any otherantigen, and advantageously is a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See, Gruber et al.,J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with aberrantCD47 signaling. For example, cysteine residue(s) can be introduced intothe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). (See Caronet al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered that hasdual Fc regions and can thereby have enhanced complement lysis and ADCCcapabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230(1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ₂₁₂Bi, ₁₃₁I, ₁₃₁In, ₉₀Y, and ₁₈₆Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against CD47

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In one embodiment, antibodies of the invention, which include amonoclonal antibody of the invention, may be used as therapeutic agents.Such agents will generally be employed to diagnose, prognose, monitor,treat, alleviate, and/or prevent a disease or pathology associated withaberrant CD47 expression, activity and/or signaling in a subject. Atherapeutic regimen is carried out by identifying a subject, e.g., ahuman patient suffering from (or at risk of developing) a disease ordisorder associated with aberrant CD47 expression, activity and/orsignaling, e.g., a cancer or other neoplastic disorder, using standardmethods. An antibody preparation, preferably one having high specificityand high affinity for its target antigen, is administered to the subjectand will generally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withthe expression, activity and/or signaling function of the target (e.g.,CD47). Administration of the antibody may abrogate or inhibit orinterfere with the binding of the target (e.g., CD47) with an endogenousligand (e.g., SIRPα) to which it naturally binds. For example, theantibody binds to the target and modulates, blocks, inhibits, reduces,antagonizes, neutralizes, or otherwise interferes with CD47 expression,activity and/or signaling.

Diseases or disorders related to aberrant CD47 expression, activityand/or signaling include, by way of non-limiting example, hematologicalcancer and/or solid tumors. Hematological cancers include, e.g.,leukemia, lymphoma and myeloma. Certain forms of leukemia include, byway of non-limiting example, acute lymphocytic leukemia (ALL); acutemyeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronicmyelogenous leukemia (CML); Myeloproliferative disorder/neoplasm (MPDS);and myelodysplasia syndrome. Certain forms of lymphoma include, by wayof non-limiting example, Hodgkin's lymphoma, both indolent andaggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicularlymphoma (small cell and large cell). Certain forms of myeloma include,by way of non-limiting example, multiple myeloma (MM), giant cellmyeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma.Solid tumors include, e.g., breast tumors, ovarian tumors, lung tumors,pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors,lung tumors, head and neck tumors, bladder tumors, esophageal tumors,liver tumors, and kidney tumors.

Symptoms associated with cancers and other neoplastic disorders include,for example, inflammation, fever, general malaise, fever, pain, oftenlocalized to the inflamed area, loss of appetite, weight loss, edema,headache, fatigue, rash, anemia, muscle weakness, muscle fatigue andabdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about100 mg/kg body weight. In some embodiments, an antibody of the inventionis administered to a subject a dose of 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 50mg/kg, 75 mg/kg, 100 mg/kg, or greater. Common dosing frequencies mayrange, for example, from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular inflammatory-relateddisorder. Alleviation of one or more symptoms of theinflammatory-related disorder indicates that the antibody confers aclinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

In another embodiment, antibodies directed against CD47 may be used inmethods known within the art relating to the localization and/orquantitation of CD47 (e.g., for use in measuring levels of CD47 and/orboth CD47 and SIRPα within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies specific to CD47, or derivative, fragment,analog or homolog thereof, that contain the antibody derived antigenbinding domain, are utilized as pharmacologically active compounds(referred to hereinafter as “Therapeutics”).

In another embodiment, an antibody specific for CD47 can be used toisolate a CD47 polypeptide, by standard techniques, such asimmunoaffinity, chromatography or immunoprecipitation. Antibodiesdirected against the CD47 protein (or a fragment thereof) can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ₁₂₅I, ₁₃₁I, ₃₅S or ₃H.

In yet another embodiment, an antibody according to the invention can beused as an agent for detecting the presence of CD47 and/or both CD47 andSIRPα protein (or a protein fragment thereof) in a sample. In someembodiments, the antibody contains a detectable label. Antibodies arepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab, scFv, or F(ab′)₂) is used. The term“labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently-labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently-labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Included within the usage of the term“biological sample”, therefore, is blood and a fraction or component ofblood including blood serum, blood plasma, or lymph. That is, thedetection method of the invention can be used to detect an analyte mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of an analyte mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of an analyte protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of an analytegenomic DNA include Southern hybridizations. Procedures for conductingimmunoassays are described, for example in “ELISA: Theory and Practice:Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) HumanPress, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T.Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and“Practice and Theory of Enzyme Immunoassays”, P. Tijssen, ElsevierScience Publishers, Amsterdam, 1985. Furthermore, in vivo techniques fordetection of an analyte protein include introducing into a subject alabeled anti-analyte protein antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

Therapeutic Administration and Formulations of CD47 Antibodies

The antibodies of the invention (also referred to herein as “activecompounds”), and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington's Pharmaceutical Sciences: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such compositions typically comprise the antibody and a pharmaceuticallyacceptable carrier. Where antibody fragments are used, the smallestinhibitory fragment that specifically binds to the binding domain of thetarget protein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.(See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993)).

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELm (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas sustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the active ingredients can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas various forms of cancer, autoimmune disorders and inflammatorydiseases. The term “in combination” in this context means that theagents are given substantially contemporaneously, either simultaneouslyor sequentially. If given sequentially, at the onset of administrationof the second compound, the first of the two compounds is preferablystill detectable at effective concentrations at the site of treatment.

For example, the combination therapy can include one or more antibodiesof the invention coformulated with, and/or coadministered with, one ormore additional therapeutic agents, e.g., one or more cytokine andgrowth factor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostaticagents, as described in more detail below. Such combination therapiesmay advantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

Preferred therapeutic agents used in combination with an antibody of theinvention are those agents that interfere at different stages in aninflammatory response. In one embodiment, one or more antibodiesdescribed herein may be coformulated with, and/or coadministered with,one or more additional agents such as other cytokine or growth factorantagonists (e.g., soluble receptors, peptide inhibitors, smallmolecules, ligand fusions); or antibodies or antigen binding fragmentsthereof that bind to other targets (e.g., antibodies that bind to othercytokines or growth factors, their receptors, or other cell surfacemolecules); and anti-inflammatory cytokines or agonists thereof.

In other embodiments, the antibodies of the invention are used asvaccine adjuvants against autoimmune disorders, inflammatory diseases,etc. The combination of adjuvants for treatment of these types ofdisorders are suitable for use in combination with a wide variety ofantigens from targeted self-antigens, i.e., autoantigens, involved inautoimmunity, e.g., myelin basic protein; inflammatory self-antigens,e.g., amyloid peptide protein, or transplant antigens, e.g.,alloantigens. The antigen may comprise peptides or polypeptides derivedfrom proteins, as well as fragments of any of the following:saccharides, proteins, polynucleotides or oligonucleotides,autoantigens, amyloid peptide protein, transplant antigens, allergens,or other macromolecular components. In some instances, more than oneantigen is included in the antigenic composition.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto CD47, the design of other therapeutic modalities beyond antibodymoieties is facilitated. Such modalities include, without limitation,advanced antibody therapeutics, such as bispecific antibodies,immunotoxins, and radiolabeled therapeutics, generation of peptidetherapeutics, gene therapies, particularly intrabodies, antisensetherapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies-one with aspecificity to CD47 and another to a second molecule that are conjugatedtogether, (ii) a single antibody that has one chain specific to CD47 anda second chain specific to a second molecule, or (iii) a single chainantibody that has specificity to CD47 and a second molecule. Suchbispecific antibodies are generated using techniques that are well knownfor example, in connection with (i) and (ii) See e.g., Fanger et al.Immunol Methods 4:72-81 (1994) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992), and in connection with (iii) See e.g.,Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing CD47.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to CD47 and antibodiesthereto, such as the antibodies of the invention or screening of peptidelibraries, therapeutic peptides can be generated that are directedagainst CD47. Design and screening of peptide therapeutics is discussedin connection with Houghten et al. Biotechniques 13:412-421 (1992),Houghten PNAS USA 82:5131-5135 (1985), Pinalla et al. Biotechniques13:901-905 (1992), Blake and Litzi-Davis BioConjugate Chem. 3:510-513(1992). Immunotoxins and radiolabeled molecules can also be prepared,and in a similar manner, in connection with peptidic moieties asdiscussed above in connection with antibodies. Assuming that the CD47molecule (or a form, such as a splice variant or alternate form) isfunctionally active in a disease process, it will also be possible todesign gene and antisense therapeutics thereto through conventionaltechniques. Such modalities can be utilized for modulating the functionof CD47. In connection therewith the antibodies of the present inventionfacilitate design and use of functional assays related thereto. A designand strategy for antisense therapeutics is discussed in detail inInternational Patent Application No. WO 94/29444. Design and strategiesfor gene therapy are well known. However, in particular, the use of genetherapeutic techniques involving intrabodies could prove to beparticularly advantageous. See e.g., Chen et al. Human Gene Therapy5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997). General designof and considerations related to gene therapeutics is also discussed inInternational Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of the CD47 molecule and itsinteractions with other molecules in accordance with the presentinvention, such as SIRPα and/or the antibodies of the invention, andothers can be utilized to rationally design additional therapeuticmodalities. In this regard, rational drug design techniques such asX-ray crystallography, computer-aided (or assisted) molecular modeling(CAMM), quantitative or qualitative structure-activity relationship(QSAR), and similar technologies can be utilized to focus drug discoveryefforts. Rational design allows prediction of protein or syntheticstructures which can interact with the molecule or specific formsthereof which can be used to modify or modulate the activity of IL-6Rc.Such structures can be synthesized chemically or expressed in biologicalsystems. This approach has been reviewed in Capsey et al. GeneticallyEngineered Human Therapeutic Drugs (Stockton Press, NY (1988)). Further,combinatorial libraries can be designed and synthesized and used inscreening programs, such as high throughput screening efforts.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with the binding of CD47 toSIRPα, or candidate or test compounds or agents that modulate orotherwise interfere with the signaling function of CD47 and/orCD47-SIRPα. Also provided are methods of identifying compounds useful totreat disorders associated with aberrant CD47 and/or CD47-SIRPαexpression, activity and/or signaling. The screening methods can includethose known or used in the art or those described herein. For example,CD47 can be immobilized on a microtiter plate and incubated with acandidate or test compound, e.g., a CD47 antibody, in the presence ofSIRPα. Subsequently, bound SIRPα can be detected using a secondaryantibody, and absorbance can be detected on a plate reader.

Methods of identifying compounds capable of promoting phagocytosis oftumor cells by macrophages are also provided. These methods can includethose known or used in the art or those described herein. For example,macrophages are incubated with labeled tumor cells in the presence of acandidate compound, e.g., a CD47 antibody. After a period of time, themacrophages can be observed for internalization of the tumor label toidentify phagocytosis. Additional details regarding these methods, e.g.,SIRPα blocking assays and phagocytosis assays, are provided in theExamples.

The invention also includes compounds identified in the screening assaysdescribed herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which modulate the signaling function of CD47. Thetest compounds of the invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. (See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates the signalingfunction of CD47 and/or the interaction between CD47 and SIRPα. Inanother embodiment, a soluble CD47 and/or both CD47 and SIRPα protein ofthe invention is provided and exposed to at least one neutralizingmonoclonal antibody. Formation of an antibody-antigen complex isdetected, and one or more candidate compounds are introduced to thecomplex. If the antibody-antigen complex is disrupted followingintroduction of the one or more candidate compounds, the candidatecompounds is useful to treat disorders associated with aberrant CD47and/or CD47-SIRPα signaling.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ₁₂₅I, ₃₅S, ₁₄C, or ₃H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a neutralizing antibody,which modulates or otherwise interferes with CD47 activity and/orsignaling.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use of either the soluble form or the membrane-bound form ofCD47 and fragments thereof. In the case of cell-free assays comprisingthe membrane-bound form of CD47, it may be desirable to utilize asolubilizing agent such that the membrane-bound form of the proteins aremaintained in solution. Examples of such solubilizing agents includenon-ionic detergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein that adds a domain that allows one or both of theproteins to be bound to a matrix can be provided. For example,GST-antibody fusion proteins or GST-antigen fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, that are thencombined with the test compound, and the mixture is incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly. Alternatively, the complexes can be dissociatedfrom the matrix, and the level of antibody-antigen complex formation canbe determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody (e.g., the 2A1 antibody, or an antibody having a variable heavychain selected from SEQ ID NOs: 5-30 and a variable light chain selectedfrom SEQ ID NOs: 31-47) or the antigen (e.g. CD47 protein) can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated antibody or antigen molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well-known withinthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, other antibodies reactive with the antibody orantigen of interest, but which do not interfere with the formation ofthe antibody-antigen complex of interest, can be derivatized to thewells of the plate, and unbound antibody or antigen trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using such other antibodiesreactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic and Prophylactic Formulations

The CD47 MAbs of the invention are used in diagnostic and prophylacticformulations. In one embodiment, a CD47 MAb of the invention isadministered to patients that are at risk of developing one or more ofthe aforementioned diseases, such as for example, without limitation,cancer or other neoplastic condition. A patient's or organ'spredisposition to one or more of the aforementioned cancers or otherneoplastic conditions can be determined using genotypic, serological orbiochemical markers.

In another embodiment of the invention, the CD47 antibody isadministered to human individuals diagnosed with a clinical indicationassociated with one or more of the aforementioned diseases, such as forexample, without limitation, cancer or other neoplastic condition. Upondiagnosis, the CD47 antibody is administered to mitigate or reverse theeffects of the clinical indication associated with one or more of theaforementioned diseases.

Antibodies of the invention are also useful in the detection of CD47and/or SIRPα in patient samples and accordingly are useful asdiagnostics. For example, the CD47 antibodies of the invention are usedin in vitro assays, e.g., ELISA, to detect CD47 and/or SIRPα levels in apatient sample.

In one embodiment, a CD47 antibody of the invention is immobilized on asolid support (e.g., the well(s) of a microtiter plate). The immobilizedantibody serves as a capture antibody for any CD47 and/or SIRPα that maybe present in a test sample. Prior to contacting the immobilizedantibody with a patient sample, the solid support is rinsed and treatedwith a blocking agent such as milk protein or albumin to preventnonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of CD47 and/or SIRPα in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using the CD47antibodies of the invention in an in vitro diagnostic assay, it ispossible to stage a disease (e.g., a clinical indication associated withischemia, an autoimmune or inflammatory disorder) in a subject based onexpression levels of CD47 and/or SIRPα. For a given disease, samples ofblood are taken from subjects diagnosed as being at various stages inthe progression of the disease, and/or at various points in thetherapeutic treatment of the disease. Using a population of samples thatprovides statistically significant results for each stage of progressionor therapy, a range of concentrations of the antigen that may beconsidered characteristic of each stage is designated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 Generation and Selection of CD47 Antibodies

CD47 antibodies were generated by immunizing mice with a recombinantprotein representing CD47-IgV (immunoglobin-like variable-type),implementing a modified rapid immunization strategy in multiple sites(Kilpatrick et al. (1997) Rapid development of affinity maturedmonoclonal antibodies using RIMMS. Hybridoma 16, 381-389). In addition,half of the mice in the immunized group received a single injection ofthe anti-mouse GITR agonist antibody, DTA-1. Following the immunizationschedule, lymph nodes from all mice (DTA-1 treated and untreated) wereharvested and dissociated, thereby enabling B-cell isolation andsubsequent fusion to a mouse myeloma cell line. Hybridoma supernatantswere screened for binding to CD47 by ELISA and by flow cytometry onDaudi (ATCC# CCL-213) cells (FIG. 1A). Hybridoma supernatants were alsoanalyzed for the ability to block the CD47-SIRPα interaction (FIG. 1B).Recombinant CD47 was immobilized on a Medisorp (NUNC) microtiter plateand subsequently incubated with the hybridoma supernatants in thepresence of recombinant human SIRPα-ECD fused to a human IgG Fc domain.Bound SIRPα was detected using an HRP conjugated anti-human IgG Fcspecific secondary antibody (Jackson Immuno Research), and absorbance at650 nm detected in plate reader.

Example 2 Characterization of CD47 Antibodies

Exemplary murine CD47 antibodies of the present invention are shown inFIG. 2. Affinity ranking of SIRPα blocking CD47 antibodies was conductedby flow cytometry on Raji (ATCC# CCL-86) (FIG. 2A) and CCRF-CEM (ATCC#CCL-119) cells (FIG. 2B). Bound CD47 antibodies were detected using aFITC conjugated anti-mouse IgG secondary antibody (JacksonImmunoResearch). The CD47 antibody known in the art, B6H12, was includedas a positive control (See, e.g., U.S. Pat. No. 5,057,604). In FIG. 2B,both the B6H12 and the 2D3, a commercially available non-SIRPα blockingantibody, were compared to antibodies generated herein. The antibodiesof the present invention display higher affinity toward the endogenous(cell surface) form of CD47 compared to the B6H12 and 2D3 antibodies.

Example 3 SIRPα Blocking Activity of CD47 Antibodies

The potency of SIRPα blocking by CD47 antibodies was measured by anELISA wherein recombinant His-tagged-CD47-IgV was immobilized on aMedisorp microtiter plate. Binding of recombinant SIRPα fused to an Fcdomain of human IgG was monitored in the presence of increasing amountsof the CD47 antibodies. Bound SIRPα was determined using an HRPconjugated anti-human IgG (Fc specific) secondary antibody (JacksonImmunoResearch). The antibodies of the present invention displayenhanced potency of SIRPα blocking compared to the B6H12 antibody. FIG.3A shows representative data of the ELISA based SIRPα blocking assay.

CD47 antibodies were analyzed by flow cytometry for their ability toblock recombinant SIRPα binding to cell surface CD47. CCRF-CEM (ATCC#CCL-119) cells were used as the source of CD47 in the assay and bindingof recombinant SIRPα fused to an Fc domain of human IgG was monitored inthe presence of increasing amounts of the CD47 antibodies. Bound SIRPαwas determined using an APC conjugated anti-human IgG (Fc specific)secondary antibody (Jackson ImmunoResearch) (FIG. 3B). B6H12 andcommercially available non-SIRPα blocking CD47 antibody 2D3 where used apositive and negative controls respectively.

Example 4 CD47 Antibody-Mediated Homotypic Interactions

SIRPα blocking CD47 antibodies were analyzed for their ability to inducecellular clustering, as known as homotypic interactions, between CD47positive cells. Daudi and Raji cells were used as candidate CD47expressing cells lines. Among the antibodies examined, the 2A1 antibodyof the present invention was the only SIRPα blocking antibody that didnot promote homotypic interactions of CD47 expressing cells.

Example 5 Hemagglutination Activity of CD47 Antibodies

One example of a homotypic interaction is hemagglutination, as evidencedby RBC aggregation. CD47 antibodies were screened for RBC agglutination,as observed by the ability of an antibody to prevent the settling ofhuman RBCs. Unexpectedly, the 2A1 antibody was found to be unique amongother CD47 antibodies for its inability to promote hemagglutination,while having high affinity and the ability to block SIRPα. Otherantibodies that displayed reduced hemagglutination did not block SIRPαbinding to CD47.

To evaluate the hemagglutinating capacity of CD47 antibodies, human RBCswere diluted to 10% in PBS and incubated at 37° C. for 2-6 hours with atitration of CD47 antibodies in a round bottom 96 well plate. Evidenceof hemagglutination is demonstrated by the presence of non-settled RBCs,appearing as a haze compared to a punctuate red dot ofnon-hemagglutinated RBCs. Unexpectedly, as shown in FIG. 4A, CD47antibodies of the invention, particularly the antibody referred toherein as 2A1, did not exhibit hemagglutinating activity. The graphshows the quantitation of the hemagglutination assay, denoted“hemagglutination index” determined by quantitating the area of the RBCpellet in the presence of the antibody, normalized to that in theabsence of the antibody.

The murine 9E4 antibody caused the most profound hemagglutination at allconcentrations tested. Thus, The 9E4 antibody binds CD47 and blocks CD47interaction with SIRP□; however, the 9E4 antibody causes profoundhemagglutination□

The VH chain region of the 9E4 antibody is provided below.

(SEQ ID NO: 78) EVQLRQSGPELVKPGASVKISCKASGYSFTDYYMYWVKQSRVRSLAWIGRINPYTGATGYDQNFKDKASLIVDKSSSTAYMELRSLTSEDSAVYYCAR GRNRYDGWFAYWGQGTLVTV 

The VL chain region of the 9E4 antibody is provided below.

(SEQ ID NO: 79) EIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLDQEDIATYFCQQGNALPPTFGG GTNLEIK 

The control antibody B6H12 caused hemagglutination as is expected forSIRPα blocking CD47 antibodies.

In order to investigate the uniqueness of the non-hemagglutinatingactivity of the 2A1 antibody, numerous other CD47 antibodies werescreened in the RBC hemagglutination assay (FIG. 4B). Included in thisassay was the chimeric version of the 2A1 antibody (2A1-xi), whichconsists of the murine variable heavy chain region of 2A1, the murinevariable light chain region of 2A1 modified at amino acid 106 (i.e.,M106I), and the constant regions of human IgG1 and human Igkappa. The VHand VL region sequences of 2A1 antibody and 2A1-xi antibody are providedin Table 1. Antibodies were tested at 12.5, 25, 50, and 100 nM.Unexpectedly, 2A1 is rare amongst the CD47 antibodies examined in FIG.4B, in that it was the only antibody in FIG. 4B with absent or reducedhemagglutinating activities. FIG. 4E shows that 2A1, chimeric 2A1(2A1-xi), and humanized variants do not cause hemagglutination.

FIG. 4C shows the results of screening additional CD47 antibodies in theRBC hemagglutination assay. As shown in FIG. 4C, the commerciallyavailable CD47 monoclonal antibody 2D3, which does not block SIRPα, didnot cause hemagglutination. However, other commercially available CD47antibodies (e.g., CC2C6, BRC126, and B6H12) which block SIRPα causedhemagglutination (FIG. 4C). Thus, prior to the invention describedherein, existing antibodies that blocked SIRPα caused hemagglutination,while existing antibodies, such as 2D3, that did not block SIRPα did notcause hemagglutination. Taken together, the antibodies of the invention(e.g., the 2A1 antibody and its humanized derivatives) are unique amongexisting CD47 antibodies in their ability to block SIRPα, but not causehemagglutination.

A high concentration range of select CD47 antibodies was retested in thehemagglutination assay (FIG. 4D). This assay revealed a pro-zone effectof hemagglutination by B6H12 and 9E4, wherein hemagglutination wasreduced at high and low ends of the concentration range tested. Thegraphical representation of the hemagglutination index also highlightsthe pro-zone effect. The pro-zone effect was also evident in FIGS. 4Cand 4E. Importantly, the mouse 2A1 and chimeric 2A1 CD47 antibodies weredevoid of hemagglutinating activity at all concentrations.

As shown in FIG. 4E, the murine 1B4 antibody displayed a narrow range ofhemagglutination.

The VH chain region of the 1B4 antibody is provided below.

(SEQ ID NO: 80) QIQLQQSGPELVKPGASVKISCKASGYTFTDYYIHWVKQRPGQGLEWIGWIYPGSGNTKYNERFKGKATLTVATSSSTAYMQLSSLTSEDTAVYFCAR REEDYFDYWGQGTLVTV 

The VL chain region of the 1B4 antibody is provided below.

(SEQ ID NO: 81) DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLTWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSY PLTFGAGTKLEIK

The hemagglutinating capacity of humanized antibodies derived from themurine 2A1 was tested as above. Importantly, the representativehumanized antibody AB6.12 in numerous human IgG isotypes (IgG1,IgG4-S228P, and IgG4-S228P/L235E) did not cause any RBChemagglutination. 2A1 and 2A1-xi were included as controls fornon-hemagglutinating antibodies, whereas B6H12 and 9E4 were included aspositive controls for hemagglutination (FIG. 4F).

Example 6 Binding to Cynomolgus Monkey CD47

The ability of murine 2A1 to bind to cynomolgus (cyno) monkey CD47 wasassessed. The B6H12 antibody has previously been reported to becross-reactive with cyno CD47 and was used as a positive control for thepresence on cyno CD47 in the assay. The experiment to measure binding of2A1 to cynomolgus monkey CD47 was designed to compare binding of 2A1 toCD47 on cynomolgus monkey B-cells and human cells, wherein the Raji cellline was used as a human CD47 positive cell. Cynomolgus peripheral bloodmononuclear cells (PBMCs) were isolated from cynomolgus whole blood byficoll-paque gradient centrifugation. Cynomolgus and human B-cells(Raji) were labeled with the human CD20 antibody ofatumumab (Arzerra) at10 μg/ml, and reacted with a dilution series of murine CD47 antibody 2A1or B6H12. B-cells labeled with human CD20 antibody were detected withpolyclonal anti-human antibody conjugated to DyLite 649, while the CD47murine antibodies were detected with polyclonal anti-mouse antibodyconjugated to DyLite 488. Cells were analyzed by flow cytometry, firstgated on live cells by FSC and SSC, then on cells positive for FL4 (CD20positive), and lastly the median FL1 (CD47 positive) was measured. Thedata were normalized by dividing the signal at each concentration by themaximum signal for each antibody on each cell population. The normalizedresults shown in FIG. 5 reveal that 2A1 does cross react with cyno CD47and has identical affinity as compared to human CD47. Consistent withthe results presented above, B6H12 had lower affinity for cell surfaceCD47 on both Raji and cynomolgus B-cells compared to antibodies of thepresent invention.

Example 7 Chimeric Antibody Generation

In order to identify the sequences of the variable regions of the heavy(VH) and light (VL) chains of the murine 2A1 antibody, ribonucleic acid(RNA) was isolated from the hybridoma and utilized in reversetranscription polymerase chain reaction (RT-PCR) (Phusion RT-PCR KitThermo Scientific) to generate first strand cDNA. A degenerative primerset that covers the complete repertoire of murine of antibody leadersequences of both VH and VL was used in a PCR wherein the first strandcDNA served as the template.

The forward primers (murine IgG leader) are provided below.

Name Sequence VH1-1 CACTGCAGGTRTCCACTCC (SEQ ID NO: 82) VH1-2CATAGCAGGTGTCCACTCC (SEQ ID NO: 83) VH1-3CRCTACAGGTGTCCACTCC (SEQ ID NO: 84) VH1-4GCYACAGMTGTCCACTCC (SEQ ID NO: 85) VH1-5CACTGCAGGTGTCCWMTCC (SEQ ID NO: 86) VH1-6CRCTRCAGGTGTKCACTCC (SEQ ID NO: 87) VH1-7GCTAWMGGTGTCCACTCC (SEQ ID NO: 88) VH1-8CCTCAGGTGTCCACTCC (SEQ ID NO: 89) VH1-9GCTACAGGTGCTCACTCC (SEQ ID NO: 90) VH1-10CACTGCAGGTGTCCTCTCT (SEQ ID NO: 91) VH1-11CAYTGCAGGTGTCCAYTGC (SEQ ID NO: 92) VH1-12GCTAMMGGTGTCCACTTC (SEQ ID NO: 93) VH1-13CTCCTGTCAKTAACTKCAGGT (SEQ ID NO: 94) VH1-14CAACTGCAGGTGTCTCTCT (SEQ ID NO: 95) VH1-15CRCTRCAGGYGTCCACTCT (SEQ ID NO: 96) VH2-1CCAAGCTGTATCCTTTCC (SEQ ID NO: 97) VH2-2CCAAGCTGTGTCCTRTCC (SEQ ID NO: 98) VH3-1CTTGACAGYCVTTCCKGGT (SEQ ID NO: 99) VH3-2CTTCACAGCCTTTCCTGGT (SEQ ID NO: 100) VH4CTTAAAAGGGGTCCAGTGT (SEQ ID NO: 101) VH5-1CAYTTTAAAARGTGTCMAGTGT (SEQ ID NO: 102) VH5-2GTTTTAAAAGGTGTCCTGTG (SEQ ID NO: 103) VH6CTYTTAAAAGGKGTCCAGWG (SEQ ID NO: 104) VH7-1CYTTTAMATGGTATCCAGTGT (SEQ ID NO: 105) VH7-2CTTTTACATGGTTTCAAGTGT (SEQ ID NO: 106) VH8GTCCCTGCATATGTCYT (SEQ ID NO: 107) VH9GATGGCAGCWGCYCAAAG (SEQ ID NO: 108) VH10CTATCAAGGTGTGCATTGT (SEQ ID NO: 109) VH11CTTTTAAAAGWTGTCCAGKGT (SEQ ID NO: 110) VH12GTGACAGTCCTTCCTGGTAG (SEQ ID NO: 111) VH14CTTCCTGATGGCAGTGGTT (SEQ ID NO: 112) VH15GCTACAGGTATCCAATCC (SEQ ID NO: 113)

The reverse primer (murine IgG constant) is provided below.

Name Sequence HC-Rev GCGTCTAGAAYCTCCACACACAGGRRCCAGTGGATAGAC (SEQ ID NO: 114)

The forward primers (murine IgKappa leader) are provided below.

Name Sequence VK1-1 CTGWTGTTCTGGATTCCTG (SEQ ID NO: 115) VK1-2GGTCAGACAGTCAGCAGT (SEQ ID NO: 116) VK2GTGCTCTGGATTCGGGAA (SEQ ID NO: 117) VK4/5-1CAGCTTCYTGCTAATCAGTG (SEQ ID NO: 118) VK4/5-2CTAATCAGTGCTTCAGGA (SEQ ID NO: 119) VK8-1GTGGGTATCTGGTRCSTGTG (SEQ ID NO: 120) VK8-2 GGAAATTTAAAAGTACCTGTGGG (SEQ ID NO: 121) VK9A/9B-1 GGTTTCMAGGTRCCAGATGT (SEQ ID NO: 122)VK9A/9B-2 CTCTGGTTYCCAGGTATC (SEQ ID NO: 123) VK10CTGTTTTCAAGGTRCCAGATGT (SEQ ID NO: 124) VK11GTTGTAATGTCCAGAGGA (SEQ ID NO: 125) VK12/13-1CTTACAGGTGCCAGATGT (SEQ ID NO: 126) VK12/13-2CTCAATTGTAGRTGCCAGATGT (SEQ ID NO: 127) VK12/13-3CACAGTAGGTGTCAGATGT (SEQ ID NO: 128) VK12/13-4GTCGTAGTTGTCAGATGT (SEQ ID NO: 129) VK12/13-5CCTCCTTCTTGGCCAAGA (SEQ ID NO: 130) VK19/28-1CTTATATGGAGCTGATGGG (SEQ ID NO: 131) VK19/28-2GTGTCTGGTGCTCATGGG (SEQ ID NO: 132) VK19/28-3CTSTGGTTGTCTGGTGTTGA (SEQ ID NO: 133) VK20GTCTCTGATTCTAGGGCA (SEQ ID NO: 134) VK21-1CTKCKCTGGGTTCCAG (SEQ ID NO: 135) VK21-2GCAGGTGTTGACGGA (SEQ ID NO: 136) VK22-1CAGGTGCCTCGTGCAC (SEQ ID NO: 137) VK22-2CTCTGGTGCCTGTGCA (SEQ ID NO: 138) VK23CTGGAYTYCAGCCTCCAGA (SEQ ID NO: 139) VK24/25-1GWTCTCTRGAGTCAGTGGG (SEQ ID NO: 140) VK24/25-2CTGGATCCCTGGAKCYACT (SEQ ID NO: 141) VK32GTTCTGCTTTTTAGGTGTG (SEQ ID NO: 142) VK33/34GATCCCAGGCATGATATGT (SEQ ID NO: 143) VK31/38CCTTCATGGTGCTCAGTGT (SEQ ID NO: 144) VKRFCCATATCAGGTGCCCAGTGT (SEQ ID NO: 145)

The reverse primer (murine IgKappa constant) is provided below.

Name Sequence LC -rev GCGTCTAGAACTGGATGGTGGGAAGATGG (SEQ ID NO: 146)

Amplified VH and VL were subsequently cloned in-frame into vectorscontaining appropriate antibody secretion sequences and human IgG1 andIgkappa constants regions, respectively, to generate murine:humanchimeric DNA constructs. These constructs were co-transfected into293Freestyle cells (Life Technologies) and the resultant antibody waspurified from the cell culture supernatant by Protein-A chromatography.To determine that the correct VH and VL sequences had been identified,the chimeric 2A1 (denoted 2A1-xi) was compared to the murine parental2A1 antibody and CD47 binding assay by flow cytometry on Raji cells(FIG. 6). B6H12 was also included as a positive control in this assay.Bound 2A1-xi was detected using a FITC-conjugated anti-human IgGsecondary antibody. Bound 2A1 and B6H12 were detected using aFITC-conjugated anti-mouse IgG secondary antibody. Apparent affinitieswere determined by non-linear fits (Prism Graphpad Software) of themedian fluorescence intensities at various antibody concentrations(Table 2). The 2A1-xi antibody has a similar binding affinity as themurine 2A1 antibody toward cell surface CD47, demonstrating that the VHand VL sequences had been properly identified.

TABLE 2 KD (apparent) (pM) Std Error R² 2A1-mIgG1 93.6 ±10.1 0.99772A1-xi 78 ±14.9 0.9922 B6H12 3786 ±310 0.9998

Example 8 Antibody Humanization

The murine 2A1 CD47 antibody was humanized to reduce the potential ofimmunogenicity when administered to human patient. The sequences of theVH and VL region of 2A1 were compared to human antibody sequences in theIMGT databank. Subsequently, a structural model was generated of the 2A1VH and VL regions using the known structures of the most closely relatedhumanized and human antibodies in the Protein Data Bank (PDB). The 3complementary determining regions (CDR) in both the heavy and lightchains of the 2A1 antibody were fixed and the murine frameworks werereplaced with numerous human frameworks that had the highest possibilityof maintaining the proper orientation of the CDRs. Constructscorresponding to each the humanized 2A1 variants were generated by genesynthesis and cloned in frame into vectors containing an appropriatesecretion sequence and human IgG1 and Igkappa constant regions. Variouscombinations of humanized heavy and light chains were co-transfected into 293Freestyle cells (Life Technologies), and resultant antibodies werepurified from the cell culture supernatant by Protein-A chromatography.

Humanized antibodies were tested for their ability to bind Raji cells byflow cytometry (FIG. 7). The 2A1-xi antibody was used as a control inmost of these assays to set the benchmark for binding affinity.Humanized antibodies were further optimized to enhance expression andreduce problematic sites including potential isomerization anddeamidation sites. An example of an optimized humanized antibody derivedfrom the murine 2A1 antibody is denoted as AB6.12 antibody, whichdisplays very similar binding affinity as the 2A1-xi antibody (FIG. 7H;Table 3). Apparent affinities were determined by non-linear fits (PrismGraphpad Software) of the median fluorescence intensities at variousantibody concentrations.

TABLE 3 KD (apparent) (pM) Std Error R2 2A1-xi 36.4 ±8.54 0.9908 AB6.1239.9 ±5.54 0.9964

The AB6.12 antibody was subsequently converted from an IgG1 to otherhuman IgG isotypes by replacing the constant domain of the heavy chain.As shown in FIG. 7I, changing the IgG isotype to a hinge stabilizedversion of IgG4 (IgG4P: S228P), and reduced Fc receptor binding variantof the hinge stabilized IgG4 (IgG4PE: S228P/L235E) did not alter bindingaffinity of the humanized antibody toward cells surface CD47 (FIG. 7I;Table 4). Apparent affinities were determined by non-linear fits (PrismGraphpad Software) of the median fluorescence intensities at variousantibody concentrations.

TABLE 4 KD (apparent) (pM) Std Error R² AB6.12-IgG1 38.6 ±10.5 0.9798AB6.12-IgG4P 35.7 ±8.4 0.9841 AB6.12-IgG4PE 34.6 ±10.9 0.9727

Throughout the humanization process, the CD47 antibodies were tested toensure the SIRPα blocking functionality was intact. As shown in FIG. 7J,multiple IgG isotypes of the humanized antibody, AB6.12, blocked theSIRPα:CD47 interaction, using the flow cytometry-based method describedabove in Example 3. Exemplary CD47 antibodies and their corresponding VHregion and VL region include those provided in Table 1.

During the humanization process, it was determined that in someembodiments, an amino acid sequence motif, “NA,” at the beginning of VHCDR3 (SEQ ID NO: 52 or SEQ ID NO: 77) is important for binding of theCD47 antibodies described herein. In some embodiments, in the absence ofamino acid residues “NA” at the beginning of VH CDR3 (SEQ ID NO: 52 orSEQ ID NO: 77), the CD47 antibodies of the invention do not bind totheir target or bind to their target with lower affinity than they wouldin the presence of amino acid residues “NA.” For example, when the “NA”motif was changed to more canonical motifs of “AR” or “AT,” binding wassubstantially reduced (i.e., greater than ten-fold). In otherembodiments, in the absence of amino acid residues “NA” at the beginningof VH CDR3 (SEQ ID NO: 52 or SEQ ID NO: 77), the CD47 antibodies of theinvention bind to their target with equivalent affinity compared tobinding in the presence of amino acid residues “NA.”

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if an amino acid substitutionin the sequences of the CD47 antibodies of the invention will result inan antibody with substantially the same function, e.g., a CD47 antibodywith the ability to block SIRPα and not cause a significant level ofhemagglutination and/or platelet depletion.

An image of the trace from size exclusion chromatography using an AKTAFLPC with a Superdex200 column is shown in FIG. 8A. The IgG1, IgG4P, andIgGPE variants of the AB6.12 antibody are shown. All three variant areover 98% monomeric. FIG. 8B is a photograph of a coomassie blue stainedSDS-PAGE gel of numerous humanized variants of 2A1 under reducing (R)and non-reducing (NR) conditions.

Example 9 CD47 Antibodies Promote Phagocytosis of Tumor Cell Lines

CD47 is a cell surface receptor that is upregulated on tumor cells andis also thought to contribute to immune evasion through its interactionwith its natural ligand SIRPα. Ligation of CD47 to SIRPα on macrophagesresults in decreased phagocytic activity. As described in detail below,it was determined if the CD47 binding and SIRPα blocking activity of the2A1 antibody, and variations thereof, promote tumor cell phagocytosis inthe presence of human macrophages.

PBMCs were isolated from human blood, and the monocytes weredifferentiated into macrophages by incubating them in AIM-V media (LifeTechnologies) for 7 days. These monocyte derived macrophages (MDMs)become adherent allowing other cells to be washed away. MDMs werescraped and re-plated in 12-well dishes and allowed to adhere for 24hours. The human tumor cell line CCRF-CEM was chosen as a target celltype because of its high CD47 expression. CCRF-CEM cells were labeledwith 0.3 μM CFSE at 37° C. for 15 minutes, then washed and added to MDMsat a ratio of 4:1 tumor cells per phagocyte, and CD47 antibody was addedat various concentrations. Phagocytosis of target cells was allowed for3 hours. Subsequently, non-phagocytosed target cells were washed awaywith PBS. The remaining phagocytes were scraped off, stained with anantibody to the macrophage marker CD14 conjugated to DyLite 649(Biolegend), and analyzed by flow cytometry. Phagocytosis was measuredby gating on live cells that were FL4 positive (CD14+), and thenassessing the percent of FL1 (CFSE+) positive cells.

FIG. 9 shows that the CD47 antibody 2A1 and its humanized variantsdemonstrated a dose-dependent increase in phagocytosis of tumor cells byMDMs. Antibody 2A1 and the humanized variant AB2.05 were unique in theirability to induce phagocytosis of tumor cells at 66.7 pM, whereas B6H12had no activity at that concentration(FIG. 9A). FIG. 9B shows how 2A1,and the humanized variants AB2.05, AB6.12-IgG1, AB6.12-IgG4P, andAB6.12-IgG4PE all induce maximal phagocytosis at 0.3 μg/ml or 2 nM,while B6H12 requires higher concentrations. This data demonstrates thatthe CD47 antibody, 2A1 (and humanized variants derived from it), inducemacrophage-mediated phagocytosis of CD47 positive tumor cells. In thisexample, CCFR-CEM cells were utilized as the CD47 positive target cell.

Example 10 Antitumor Activity of CD47 Antibodies

The anti-tumor activity of the murine CD47 antibodies was evaluated in aRaji model of lymphoma. Raji cells were implanted subcutaneously inNOD/SCID mice, and randomized into 5 groups (10 mice per group, day 0).Group 1: Vehicle (buffer only); Group 2: B6H12 (positive control); Group3: 1B4; Group 4: 2A1; and Group 5: 9E4. Treatment with each antibody orvehicle (buffer only) began when tumors were palpable (50 mm3, day 13)and mice were euthanized when their tumor volumes reached ˜1500 mm3.Tumor volumes were measured 3 times per week. Antibodies were dosedintravenously (IV) with 200 μg 3 times per week for 3 weeks (9 totaldoses per mouse). Treatment started on day 13 and ended on day 32.

As shown in FIG. 10A, CD47 antibodies of the invention, particularly the2A1 antibody, demonstrated anti-tumor activity in this animal model oflymphoma. To reach a tumor volume of 1500 mm3, Group 1 (vehicle only)required ˜25 days; Group 2 (B6H12.2) required ˜45 days; Group 3 (1B4)required ˜37 days; Group 4 (2A1) required ˜85 days; and Group 5 (9E4)required ˜40 days to reach a tumor volume ˜1500 mm3. These data indicatethat antibody 2A1 was significantly more potent than all CD47 bindingantibodies tested, including B6H12 that was known to bind CD47, blockCD47 interaction with SIRPα, and suppress tumor formation in mousemodels of human cancer. Unexpectedly, tumor suppression activity ofthese CD47 antibodies did not correlate with their potency of bindingCD47 or blocking CD47 interaction with SIRPα, which would be expectedbased upon published data.

As described in Examples 2 and 3, 2A1, 1B4, and 9E4 had similar affinityfor CD47 and similar potency for blocking CD47 interaction with SIRPα.In addition, the enhanced efficacy of 2A1 cannot be explained bydifferences in the Fc domain of the antibodies described since allantibodies used in this study were comprised of identical mouse IgG1domains. Thus, in addition to unique composition of matter, the 2A1antibody possesses unexpected and unique characteristics including theinability to induce homotypic interactions between CD47 expressingcells, e.g., red blood cells, and enhanced tumor suppression activitythat cannot be explained by enhanced binding to CD47 or an enhancedability to block CD47 interaction with SIRPα.

To confirm that the humanized 2A1 antibodies maintained their anti-tumoractivity, a similar Raji tumor study was conducted. The study design wasthe same as described above. Raji cells were implanted subcutaneously inNOD/SCID mice and randomized into 5 groups (10 mice per group, day 0).In this study, antibodies were dosed intraperitoneal (IP) with 200 μg 3times per week for 3 weeks (9 total doses per mouse), and tumor volumeswere measured 3 times per week. However, for this study, the mouse IgG12A1 antibody (group 2) was compared to a humanized derivative, AB6.12.For this study, AB6.12 was constructed (as described in EXAMPLE 8) intohuman IgG1 (Group 3), human IgG4P (Group 4) and human IgG4PE (Group 4).Thus, this experiment was designed to address the influence of 2A1humanization on its tumor suppression activity and the potential role ofFc domain effector function that is known in the art to contribute tothe antitumor activities of many antibodies. It has been well documentedthat human IgG1 possesses significantly more effector function comparedto human IgG4P. IgG4PE was developed to further reduce effectorfunction. As can be seen in FIG. 10B, humanization of 2A1 did notdiminish the antitumor activity of 2A1, and in fact may have enhancedit. AB6.12-hIgG1, AB6.12-hIgG4P, and AB6.12-hIgG4PE all showed similaranti-tumor activity that appears significantly greater than mouse 2A1(2A1mIgG). This result is unexpected since 2A1mIgG1, AB6.12-hIgG1,AB6.12-hIgG4P and AB6.12-hIgG4PE have similar CD47 binding and SIRPαblocking activities. In addition, since AB6.12-hIgG1, AB6.12-hIgG4P andAB6.12-hIgG4PE have similar anti-tumor activities, it appears thateffector function does play a role in the efficacy of the humanized 2A1antibody AB6.12.

Example 11 Co-Crystallization of CD47 Antibodies with CD47

CD47 is 5 pass transmembrane protein with a single extracellular IgV(immunoglobin-like variable-type) domain that is highly glycosylated at6 sites. The structure of the CD47-IgV domain has been solved in complexwith the IgV domain of SIRPα, its natural ligand (Protein Data Bank(PDB) Reference No. 2JJS; Hatherley et al., 2008 Mol Cell, 25; 31(2):266-77 (FIG. 11A)). The structure shows SIRPα-IgV binding to CD47-IgV onan apical epitope including the N-terminal pyroglutamate of CD47. Thisstructure sufficiently explains how both cell surface transmembraneproteins can productively interact from adjacent cells in a head to headorientation. The X-ray crystallographic structure of CD47-IgV in complexwith the B6H12 Fab is presented in FIG. 11B. For clarity, the constantregions of the Fab (CH1 and CL) were omitted in the Figure, and only theFv (VH and VL) is presented. This revealed an apical binding site,positioning this antibody on a surface extremely distal from the cellmembrane (FIG. 11B). The mechanism of SIRPα blocking by B6H12 isapparent from this structure. The orientation purposes relative locationof the cell membrane is depicted as a dashed line in FIG. 11.

In order to determine the target epitope of the antibodies of thepresent invention, the X-ray crystallographic structure of theco-complex of CD47-IgV domain and the Fab of 2A1-xi (chimeric antibodywith human CH1 and CL domains) was determined (FIG. 11C). For clarity,the constant regions of the Fab (CH1 and CL) were omitted in the Figure,and only the Fv (VH and VL) is presented. Unlike the previouslydetermined structure of CD47 binding SIRPα in a head to head orientation(FIG. 11A), and the B6H12 antibody being positioned apically away fromthe membrane (FIG. 11B), the structure of 2A1 in complex with CD47revealed binding of the antibody to CD47 near the membrane in anunexpected and unique head to side orientation (FIG. 11C). The 2A1epitope on CD47 is discontinuous, and includes residues Y37, K39, K41,the KGRD (SEQ ID NO: 56) loop (residues 43-46), D51, H90, N93, E97, T99,E104, and E106 of CD47 when numbered in accordance with SEQ ID NO: 147(i.e., SEQ ID NO: 48 excluding the signal sequence (amino acids 1-18)).The structure of 2A1 bound to CD47 also reveals that the VH is primarilyinvolved in binding to the KGRD (SEQ ID NO: 56) loop of CD47, while theVK domain interacts with apical residues including Y37, T102, and E104,which are involved in SIRPα binding. Therefore, it is primarily the VKdomain that physically precludes SIRPα binding to CD47. These structuralstudies suggest that the unique epitope which 2A1 binds to is on theside of CD47. In contrast to CD47 antibodies known in the art, theorientation of the 2A1 VH region in a membrane proximal position arecritical features of this antibody that prevent a significant level ofred blood cell hemagglutination by constraining the antibody such thatit cannot bridge to CD47 molecules on adjacent cells.

Example 12 Effect of Isotype and Isotype Mutations on Platelet Depletion

The primary Fc dependent functions of an antibody (e.g., a CD47antibody) for target cell elimination are complement dependentcytotoxicity (CDC) initiated by binding C1q to the Fc region; antibodydependent cytotoxicity (ADCC) mediated by the interaction of the Fcregion with Fcγ receptors (FcγRs), primary FcγRIIIa on immune effectorcells (e.g., NK cells and Neutrophils); and antibody dependent cellularphagocytosis (ADCP) which is carried out by macrophages through therecognition of opsinized target cells via FcγRI. Antibody subclasseshave differences in their abilities to mediate Fc-dependent effectoractivities. In humans the IgG1 and IgG3 subclasses have the high potencyfor CDC due to binding C1q. In addition the IgG1 subclass has thehighest affinity for FcγRs and is thereby the most potent in terms ofADCC and Fc-dependent ADCP. The IgG4 subclass is devoid of C1q bindingability and has greatly reduced FcγR binding affinity and thereby hassignificantly diminished effector function.

The effect of antibodies that bind CD47 on platelet depletion wasinvestigated. Treatment of a cynomolgus monkey with a single dose of anantibody of the IgG1 subclass that binds to CD47 resulted in significantdepletion of platelets at all doses tested (10, 30, 100 mg/kg) (FIG.12C-D), compared to no significant depletion of platelets when vehiclewas administered (FIG. 12A-B). Thus, antibodies of the IgG1 subclassthat bind CD47 can result in the depletion of platelets in aFc-dependent manner.

To determine whether a different subclass of antibody also causesplatelet depletion, the experiment was repeated with a CD47 antibody ofthe IgG4 subclass. The IgG4 subclass of antibody that binds CD47 (IgG4P,with the mutation S228P to stabilize the hinge region of the antibody)also resulted in depletion of platelets at all concentrations tested,albeit to a lesser degree relative to the IgG1 subclass version (FigureE-F). Next, a mutant form of the IgG4 subclass of anti-CD47 antibody(IgG4PE, with the S228P mutation as well as a L235E mutation to reduceFcγR binding) was tested for platelet depletion (FIG. 12G-H).Surprisingly, the IgG4PE antibody did not result in depletion ofplatelets even at very high (100 mg/kg) doses. Thus, a CD47 bindingantibody with severely reduced FcγR binding and effector function doesnot result in platelet depletion.

Example 13 Red Blood Cell (RBC) Depleting Activity of the CD47Antibodies

Weiskopf et al found that when a CD47 antibody which bound mouse CD47 oran affinity evolved SIRPα-Fc fusion protein was administered to miceand/or cynomolgus monkeys, red blood cell loss and anemia were observed.(See, Weiskopf et al. Engineered SIRPα Variants as ImmunotherapeuticAdjuvants to Anticancer Antibodies; Science 2013; 341:88). Prior to theinvention presented herein, all known CD47 binding molecules (e.g., CD47antibodies and recombinant SIRPα-Fc fusion proteins) that block SIRPαand contain an Fc domain also induced RBC depletion.

Experiments were performed to determine the effect of the SIRPαblocking, non-hemagglutinating CD47 antibodies of the invention on redblood cell depletion in vivo. Surprisingly, non-hemagglutinating CD47antibodies of the invention do not cause significant red blood celldepletion after administration. Specifically, the IgG4-P and IgG4-PEvariants of the AB06.12 antibody were given to cynomolgus monkeys atdoses of 10, 30, and 100 mg/kg via intravenous infusion. Three monkeyswere used per dose group for each antibody. Red blood cell counts weremonitored over time and compared to vehicle treated monkeys. FIG. 13depicts the mean RBC counts from antibody-treated monkeys, normalized tothe mean RBC counts of the vehicle-treated monkeys. No significant RBCdepletion in the antibody treated monkeys was observed compared tovehicle treated animals, demonstrating that non-hemagglutinating CD47can be administered at high doses and without inducing amenia in thesubject.

What is claimed is:
 1. An isolated monoclonal antibody that binds toCD47 or an immunologically active fragment thereof, wherein the antibodydoes not cause a significant level of red blood cell depletion, anemia,or both red blood cell depletion and anemia after administration.
 2. Theisolated monoclonal antibody of claim 1, wherein the antibody does notcause a significant level of platelet depletion after administration. 3.The isolated monoclonal antibody of claim 1, wherein the antibody doesnot cause a significant level of agglutination of cells afteradministration.
 4. The antibody of claim 1, wherein the antibody ischimeric, humanized, or fully human.
 5. The antibody of claim 1, whereinthe CD47 is human CD47.
 6. The antibody of claim 1, wherein the antibodyor immunologically active fragment thereof prevents CD47 frominteracting with signal-regulatory-protein α (SIRPα).
 7. The antibody ofclaim 6, wherein the antibody or immunologically active fragment thereofpromotes macrophage-mediated phagocytosis of a CD47-expressing cell. 8.The antibody of claim 1, wherein the antibody or immunologically activefragment thereof is an IgG isotype selected from the group consisting ofIgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype.
 9. Theantibody of claim 1, wherein the antibody or immunologically activefragment thereof comprises a variable heavy (VH) chain region selectedfrom the group consisting of SEQ ID NOs: 5-30.
 10. The antibody of claim1, wherein the antibody or immunologically active fragment thereofcomprises a variable light (VL) chain region selected from the groupconsisting of SEQ ID NOs: 31-47.
 11. The antibody of claim 1, whereinthe antibody or immunologically active fragment thereof comprises a VHregion provided in any one of SEQ ID NOs: 5-30 and a VL region providedin any one of SEQ ID NOs: 31-47.
 12. The antibody of claim 11, whereinthe antibody or immunologically active fragment thereof comprises a VHregion provided in any one of SEQ ID NOs: 5, 7, 8, 11, 12, 15-17, 20-22,and 27-30 paired with a VL region provided in any one of SEQ ID NOs: 31,32, 35, 40, 41, 42, 43, 44, and
 47. 13. The antibody of claim 11,wherein the antibody comprises a combination of a VH chain region and aVL chain region selected from the combinations listed in Table
 1. 14.The antibody of claim 1, wherein the antibody or immunologically activefragment thereof comprises a VH complementarity determining region 1(CDR1) sequence set forth in SEQ ID NO: 50, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ IDNO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66, a VH CDR2sequence set forth in SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 76, a VH CDR3 sequence set forthin SEQ ID NO: 52 or SEQ ID NO: 77, a VL CDR1 sequence set forth in SEQID NO: 53, SEQ ID NO: 67, or SEQ ID NO: 68, a VL CDR2 sequence set forthin SEQ ID NO: 54, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71 and aVL CDR3 sequence set forth in SEQ ID NO:
 55. 15. The antibody of claim14, wherein the antibody or immunologically active fragment thereofcomprises a VH CDR1 sequence set forth in SEQ ID NO: 50, a VH CDR2sequence set forth in SEQ ID NO: 51, a VH CDR3 sequence set forth in SEQID NO: 52, a VL CDR1 sequence set forth in SEQ ID NO: 53, a VL CDR2sequence set forth in SEQ ID NO: 54, and a VL CDR3 sequence set forth inSEQ ID NO:
 55. 16. The antibody of claim 14, wherein the antibody orimmunologically active fragment thereof comprises a VH CDR1 sequence setforth in SEQ ID NO: 50, a VH CDR2 sequence set forth in SEQ ID NO: 72, aVH CDR3 sequence set forth in SEQ ID NO: 52, a VL CDR1 set forth in SEQID NO: 53, a VL CDR2 sequence set forth in SEQ ID NO: 71, and a VL CDR3sequence set forth in SEQ ID NO:
 55. 17. The antibody of claim 1,wherein the antibody binds to CD47 in a head to side orientation,wherein a VH chain of the antibody is positioned near the membrane of aCD47 expressing cell, and wherein a VL chain of the antibody occludes aSIRPα binding site on CD47.
 18. The antibody of claim 1, wherein theantibody binds to CD47 in a head to side orientation, wherein a VL chainof the antibody is positioned near the membrane of a CD47 expressingcell, and wherein a VH chain of the antibody occludes a SIRPα bindingsite on CD47.
 19. The antibody of claim 1, wherein the antibody binds toa discontinuous epitope on CD47.
 20. The antibody of claim 19, whereinthe antibody binds to a CD47 loop comprising SEQ ID NO:
 56. 21. Theantibody of claim 19, wherein the discontinuous epitope comprises aminoacids residues Y37, K39, K41, K43, G44, R45, D46, D51, H90, N93, E97,T99, E104, or E106 of CD47 when numbered in accordance with SEQ ID NO:147.
 22. The antibody of claim 3, wherein the antibody does not cause asignificant level of hemagglutination of red blood cells afteradministration.
 23. The antibody of claim 1, wherein the antibody orimmunologically active fragment thereof is an IgG isotype selected fromIgG4P and IgG4PE.
 24. A pharmaceutical composition comprising theantibody of claim 1 or an immunologically active fragment thereof and acarrier.
 25. A method of alleviating a symptom of a cancer or otherneoplastic condition, the method comprising administering the antibodyof claim 1 or an immunologically active fragment thereof to a subject inneed thereof in an amount sufficient to alleviate the symptom of thecancer or other neoplastic condition in the subject.
 26. The method ofclaim 25, wherein the subject is a human.
 27. The method of claim 25,wherein the antibody is chimeric, humanized, or fully human.
 28. Themethod of claim 25, wherein the CD47 is human CD47.
 29. The method ofclaim 25, wherein the antibody or immunologically active fragmentthereof prevents CD47 from interacting with SIRPα.
 30. The method ofclaim 25, wherein the antibody or immunologically active fragmentthereof is an IgG isotype selected from the group consisting of IgG1isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype.
 31. The method ofclaim 25, wherein the antibody or immunologically active fragmentthereof is an IgG isotype selected from IgG4P and IgG4PE.
 32. The methodof claim 25, further comprising administering chemotherapy.
 33. Themethod of claim 32, wherein said chemotherapy is radiotherapy.
 34. Themethod of claim 25, wherein the antibody is administered to the subjectat a dose of at least 10 mg/kg.
 35. The method of claim 25, wherein theantibody is administered to the subject at a dose of at least 30 mg/kg.